Pyrazolo and triazolo bicyclic compounds as jak kinase inhibitors

ABSTRACT

The invention provides compounds of formula (I): 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically-acceptable salt thereof, wherein the variables are defined in the specification, that are inhibitors of JAK kinases, particularly JAK3. The invention also provides crystalline forms, pharmaceutical compositions comprising such compounds, methods of using such compounds to treat gastrointestinal and other inflammatory diseases, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/539,642, filed on Aug. 1, 2017, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to pyrazolo and triazolo bicyclic compoundsuseful as JAK kinase inhibitors and more particularly as JAK3 inhibitorsthat are selective for JAK3 over other members of the JAK kinase familysuch as JAK1, JAK2 and TYK2. The invention is also directed tocrystalline forms, pharmaceutical compositions comprising suchcompounds, methods of using such compounds to treat inflammatorydiseases, and processes and intermediates useful for preparing suchcompounds.

State of the Art

Ulcerative colitis is a chronic inflammatory disease of the colon. Thedisease is characterized by inflammation and ulceration of the mucosallayer of the rectum and the large intestine. Common symptoms includediarrhea, bloody stools, and abdominal pain.

The clinical course is intermittent, marked by alternating periods ofexacerbation and remission. Incidence seems to be greater in developedthan in developing countries. An estimated 1.2 million people in majorindustrialized countries suffer from ulcerative colitis and the numbersare expected to increase along with population growth. Patients withulcerative colitis are at an increased risk of developing colorectalcancer. (e.g. Danese et al. N Engl J Med, 2011, 365, 1713-1725).Although there exists a variety of therapeutic options to promote andmaintain remission of ulcerative colitis (UC) in patients, none isideal. There remains an unmet medical need for an effective therapy topromote and maintain remission of moderate to severe UC without thesafety concerns resulting from chronic, systemic immunosuppression.

Although the precise pathogenesis of UC is unclear, it is apparent thatproinflammatory cytokines play a pivotal role in the immunologicalresponse (Strober et al., Gastroenterol, 2011, 140, 1756-1767). Many ofthe proinflammatory cytokines most commonly elevated in UC (e.g., IL-4,IL-6, IL-13, IL-15, IL-23, IL-24, IFNγ and leptin), rely on the JAKfamily of tyrosine kinases (i.e., JAK1, JAK2, JAK3 and Tyk2) for signaltransduction.

Inhibition of the JAK3 enzyme blocks the signaling of many keypro-inflammatory cytokines. Thus JAK3 inhibitors are likely to be usefulin the treatment of ulcerative colitis and other gastrointestinalinflammatory diseases such as Crohn's disease and immune checkpointinhibitor induced colitis. JAK3 inhibitors are also likely to be usefulfor the treatment of inflammatory skin diseases such as atopicdermatitis and inflammatory respiratory disorders such as allergicrhinitis, asthma, and chronic obstructive pulmonary disease (COPD). Inaddition, JAK3 inhibitors may also be useful in the treatment of manyocular diseases for which inflammation plays a prominent role such asuveitis, diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, retinal vein occlusion (RVO) andatopic keratoconjunctivitis.

Selectivity for JAK3 over JAK1 is anticipated to be beneficial as thereis evidence that JAK3 selectivity allows sparing of potentiallybeneficial cytokines such as IL-10 which has been involved in mucosalhealing, IL-22 which is involved in mucus barrier protection andepithelial regeneration, and IL-6 which is involved in the proliferationof intestinal epithelial cells. Selectivity for JAK3 over JAK2 alsoallows sparing of erythropoietin (EPO) and thrombopoietin (TPO)signaling. Therefore, it would be desirable to provide new compoundswhich are selective JAK3 inhibitors over other members of the JAK kinasefamily such as JAK1, JAK2 and TYK2.

Finally, due to the modulating effect of the JAK/STAT pathway on theimmune system, systemic exposure to JAK inhibitors may have an adversesystemic immunosuppressive effect. It would be desirable, therefore, toprovide new JAK3 inhibitors which have their effect at the site ofaction without significant systemic effects.

In particular, for the treatment of gastrointestinal inflammatorydiseases, such as ulcerative colitis, it would be desirable to providenew JAK3 inhibitors which can be administered orally and achievetherapeutically relevant exposure in the gastrointestinal tract withminimal systemic exposure. For skin diseases, it would be desirable toprovide new JAK3 inhibitors that could be administered topically to theskin with minimal systemic exposure.

Therefore, it would be desirable to provide new compounds which areselective JAK3 inhibitors over other members of the JAK kinase familysuch as JAK1, JAK2 and TYK2, and have minimal systemic exposure.

SUMMARY OF THE INVENTION

In one aspect, the invention provides novel compounds having activity asJAK kinase inhibitors and more particularly as JAK3 inhibitors.

Accordingly, the invention provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

X¹ and X² are each independently selected from N and CH;

X³ is selected from the group consisting of N, CH, C—CH₃, C—CF₃, C—CHF₂,C—CH₂—O—CH₃, C—SMe, C—NMe₂, C—NH—CH₃, C—Cl, C—CN, and C—OMe;

is selected from the group consisting of

R^(a), R^(b), R^(c), and R^(f) are each independently selected from thegroup consisting of H and C₁₋₃ alkyl;

R^(d), R^(e), R^(g), R^(h), R^(i), R^(j), R^(m), R^(n) and R^(o) areeach independently selected from the group consisting of H and C₁₋₃alkyl wherein the C₁₋₃ alkyl group may be optionally substituted with 1to 3 halogens;

Optionally R^(d) and R^(e) may be joined to form a cyclopropyl ring;

A is selected from the group consisting of

(a) a 4 to 10 membered monocyclic heterocyclic group containing onenitrogen atom and optionally containing one additional heteroatomselected from N, S, S(O)₂ and O, and

(b) a 6 to 10 membered multicyclic heterocyclic group containing onenitrogen atom and optionally containing one additional heteroatomselected from N, S, and O, wherein L is linked to a carbon atom in A andA is optionally substituted with 1 to 3 R^(k) groups;

each R^(k) is independently selected from the group consisting of F, CN,C₁₋₃ alkoxy, cyclopropyl, and C₁₋₃ alkyl, wherein the C₁₋₃ alkyl groupmay be optionally substituted with OH, OMe or 1 to 3 halogens;

R¹ is selected from the group consisting of

wherein R^(p) and R^(q) are each independently selected from the groupconsisting of H, C₃₋₅ cycloalkyl and C₁₋₆ alkyl, wherein the C₁₋₆ alkylgroup may be optionally substituted with 1 to 3 substituentsindependently selected from the group consisting of C₁₋₃ alkoxy and—S—C₁₋₃ alkyl,

or R^(p) and R^(q) form a 4 to 6 membered monocyclic heterocyclic groupcontaining one nitrogen atom and optionally containing one additionalheteroatom selected from N, S, and O, wherein the 4 to 6 memberedmonocyclic heterocyclic group is optionally substituted with 1 to 3substituents independently selected from the group consisting of C₁₋₆alkyl, C₁₋₃ alkoxy, —S—C₁₋₃ alkyl and —C₁₋₃ alkyl-C₁₋₃ alkoxy;

R² is selected from the group consisting of H, Cl, OMe, Me and F;

R³ is selected from the group consisting of H and F;

R⁴ is selected from the group consisting of H and F; and

R⁵ is selected from the group consisting of H, Me and F.

The disclosure also provides some crystalline forms of certaincompounds, Form 1, Form 2, Form 2b, Form 3, and Form 4.

The invention also provides a pharmaceutical composition comprising acompound of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form of the disclosure, and apharmaceutically-acceptable carrier.

The invention also provides a method of treating gastrointestinalinflammatory disease, in particular, ulcerative colitis, in a mammal,the method comprising administering to the mammal a compound of thedisclosure, or a pharmaceutically acceptable salt thereof, or acrystalline form of the disclosure, or a pharmaceutical composition ofthe disclosure.

The invention also provides a method of treating inflammatory diseasesor disorders of the skin in a mammal, the method comprising applying acompound of the disclosure, or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition of the disclosure to the skinof the mammal.

The invention also provides a method of treating cutaneous T-celllymphoma in a mammal, the method comprising applying a pharmaceuticalcomposition comprising a compound of the disclosure, or apharmaceutically acceptable salt thereof, to the skin of the mammal.

In another aspect, the invention also provides a process describedherein, which is useful for preparing the compounds of the disclosure.

The invention also provides a compound of the disclosure or apharmaceutically acceptable salt thereof, as described herein for use inmedical therapy, as well as the use of a compound of the disclosure, ora pharmaceutically acceptable salt thereof, or a crystalline formthereof, in the manufacture of a formulation or medicament for treatinga gastrointestinal inflammatory disease, or an inflammatory disease ofthe skin in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of crystallineForm 1 of compound 3 (hereinafter Form 1).

FIG. 2 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form 1.

FIG. 3 shows a thermal gravimetric analysis (TGA) plot of crystallineForm 1.

FIG. 4 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm 1 observed at a temperature of about 25° C.

FIG. 5 shows a powder x-ray diffraction (PXRD) pattern of crystallineForm 2 of compound 3 (hereinafter Form 2).

FIG. 6 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form 2.

FIG. 7 shows a thermal gravimetric analysis (TGA) plot of crystallineForm 2.

FIG. 8 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm 2 observed at a temperature of about 25° C.

FIG. 9 shows a powder x-ray diffraction (PXRD) pattern of crystallineForm 3 of compound 1 (hereinafter Form 3).

FIG. 10 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form 3.

FIG. 11 shows a thermal gravimetric analysis (TGA) plot of crystallineForm 3.

FIG. 12 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm 3 observed at a temperature of about 25° C.

FIG. 13 shows a powder x-ray diffraction (PXRD) pattern of crystallineForm 4 of compound 1 (hereinafter Form 4).

FIG. 14 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form 4.

FIG. 15 shows a thermal gravimetric analysis (TGA) plot of crystallineForm 4.

FIG. 16 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm 4 observed at a temperature of about 25° C.

FIG. 17 shows a powder x-ray diffraction (PXRD) pattern of a dehydratedcrystalline form of compound 3 (hereinafter Form 2b).

DETAILED DESCRIPTION OF THE INVENTION

Among other aspects, the invention provides JAK kinase inhibitors offormula (I) which are selective for JAK3 over other members of the JAKkinase family such as JAK1, JAK2 and TYK2, pharmaceutically-acceptablesalts thereof, and intermediates for the preparation thereof.

In one aspect, the invention provides novel compounds having activity asJAK kinase inhibitors, particularly as JAK3 kinase inhibitors.

Accordingly, the invention provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

X¹ and X² are each independently selected from N and CH;

X³ is selected from the group consisting of N, CH, C—CH₃, C—CF₃, C—CHF₂,C—CH₂—O—CH₃, C—SMe, C—NMe₂, C—NH—CH₃, C—Cl, C—CN, and C—OMe;

is selected from the group consisting of

R^(a), R^(b), R^(c), and Ware each independently selected from the groupconsisting of H and C₁₋₃ alkyl;

R^(d), R^(e), R^(g), R^(h), R^(i), R^(j), R^(l), R^(m), R^(n) and R^(o)are each independently selected from the group consisting of H and C₁₋₃alkyl wherein the C₁₋₃ alkyl group may be optionally substituted with 1to 3 halogens; optionally R^(d) and R^(e) may be joined to form acyclopropyl ring;

A is selected from the group consisting of

(a) a 4 to 10 membered monocyclic heterocyclic group containing onenitrogen atom and optionally containing one additional heteroatomselected from N, S, S(O)₂ and O, and

(b) a 6 to 10 membered multicyclic heterocyclic group containing onenitrogen atom and optionally containing one additional heteroatomselected from N, S, and O, wherein L is linked to a carbon atom in A andA is optionally substituted with 1 to 3 R^(k) groups;

each R^(k) is independently selected from the group consisting of F, CN,C₁₋₃ alkoxy, cyclopropyl, and C₁₋₃ alkyl, wherein the C₁₋₃ alkyl groupmay be optionally substituted with OH, OMe or 1 to 3 halogens;

R¹ is selected from the group consisting of

wherein R^(p) and R^(q) are each independently selected from the groupconsisting of H, C₃₋₅ cycloalkyl and C₁₋₆ alkyl, wherein the C₁₋₆ alkylgroup may be optionally substituted with 1 to 3 substituentsindependently selected from the group consisting of C₁₋₃ alkoxy and—S—C₁₋₃ alkyl,

or R^(p) and R^(q) form a 4 to 6 membered monocyclic heterocyclic groupcontaining one nitrogen atom and optionally containing one additionalheteroatom selected from N, S, and O, wherein the 4 to 6 memberedmonocyclic heterocyclic group is optionally substituted with 1 to 3substituents independently selected from the group consisting of C₁₋₆alkyl, C₁₋₃ alkoxy, —S—C₁₋₃ alkyl and —C₁₋₃ alkyl-C₁₋₃ alkoxy;

R² is selected from the group consisting of H, Cl, OMe, Me and F;

R³ is selected from the group consisting of H and F;

R⁴ is selected from the group consisting of H and F; and

R⁵ is selected from the group consisting of H, Me and F.

In some embodiments, X³ is CH.

In some embodiments, R^(p) and R^(q) are each independently selectedfrom the group consisting of H, cyclobutyl and C₁₋₄ alkyl, wherein theC₁₋₄ alkyl group may be optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of C₁₋₂ alkoxy and—S—C₁₋₂ alkyl,

or R^(p) and R^(q) form a 4 to 6 membered monocyclic heterocyclic groupcontaining one nitrogen atom and optionally containing one additionalheteroatom selected from S, and O, wherein the 4 to 6 memberedmonocyclic heterocyclic group is optionally substituted with 1 or 2substituents independently selected from the group consisting of C₁₋₃alkyl, C₁₋₂ alkoxy, —S—C₁₋₂ alkyl and —C₁₋₃ alkyl-C₁₋₂ alkoxy.

In some embodiments, R^(p) and R^(q) are each independently selectedfrom the group consisting of H, cyclobutyl and C₁₋₄ alkyl, wherein theC₁₋₄ alkyl group may be optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of OMe and —SEt,

or R^(p) and R^(q) form a 4 to 6 membered monocyclic heterocyclic groupcontaining one nitrogen atom and optionally containing one additionalheteroatom selected from S, and O, wherein the 4 to 6 memberedmonocyclic heterocyclic group is optionally substituted with 1 or 2substituents independently selected from the group consisting of Me,OMe, CH₂OMe, and —SMe.

In some embodiments, R¹ is selected from the group consisting of

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

-   -   The 6 to 10 membered multicyclic heterocyclic group may be        spiro-cyclic, fused and/or bridged.    -   In some embodiments, the 6 to 10 membered multicyclic        heterocyclic group is a spiro-cyclic heterocyclic group. In some        embodiments, the 6 to 10 membered multicyclic heterocyclic group        is a fused heterocyclic group. In some embodiments, the 6 to 10        membered multicyclic heterocyclic group is a bridged        heterocyclic group.    -   In some embodiments, A is selected from the group consisting of

(a) a 4 to 8 membered monocyclic heterocyclic group containing onenitrogen atom and optionally containing one additional heteroatomselected from N, S, S(O)₂ and O, and

(b) a 6 to 10 membered multicyclic heterocyclic group containing onenitrogen atom and optionally containing one additional heteroatomselected from N, S, and O,

wherein L is linked to a carbon atom in A and A is optionallysubstituted with 1 to 3 R^(k) groups.

In some embodiments, A is selected from the group consisting of

(a) a 4 to 6 membered monocyclic heterocyclic group containing onenitrogen atom and optionally containing one additional heteroatomselected from N, S, S(O)₂ and O, and

(b) a 6 to 10 membered multicyclic heterocyclic group containing onenitrogen atom and optionally containing one additional heteroatomselected from N, S, and O, wherein L is linked to a carbon atom in A andA is optionally substituted with 1 to 3 R^(k) groups.

In some embodiments, A is selected from the group consisting of:

-   -   (a) a 4 to 6 membered monocyclic heterocyclic group containing        one nitrogen atom and optionally containing one additional        heteroatom selected from N, S, S(O)₂ and O, and    -   (b) a 7 or 8 membered multicyclic heterocyclic group containing        one nitrogen atom and optionally containing one additional        heteroatom selected from N, S and O,

wherein L is linked to a carbon atom in A and A is optionallysubstituted with 1 to 3 R^(k) groups.

-   -   In some embodiments, A is selected from the group consisting of        azetidine, pyrrolidine, piperidine, morpholine,        2-azaspiro[3.3]heptane, thiomorpholine and nortropane.

In some embodiments,

is selected from the group consisting of

In some embodiments,

is selected from the group consisting of:

In some embodiments, X¹ and X² are both CH. In some embodiments, X¹ is Nand X² is CH. In some embodiments, X¹ and X² are both N. In someembodiments, X¹ is CH and X² is N.

In some embodiments, R² is H. In some embodiments, R² is F. In someembodiments, R² is Cl. In some embodiments, R² is OMe. In someembodiments, R² is Me. In some embodiments, R³ is H. In someembodiments, R³ is F. In some embodiments, R⁴ is H. In some embodiments,R⁴ is F. In some embodiments, R⁵ is H. In some embodiments, R⁵ is F. Insome embodiments, R⁵ is Me.

In some embodiments,

is selected from the group consisting of:

In some embodiments

is selected from the group consisting of:

The invention also provides a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein

X¹ and X² are both CH, or X¹ and X² are both N, or X¹ is N and X² is CH;

is selected from the group consisting of

R^(c), R^(d), and R^(e) are each independently selected from the groupconsisting of H and methyl;

A is selected from the group consisting of azetidine, pyrrolidine,piperidine, and morpholine;

wherein L is linked to a carbon atom in A and A is optionallysubstituted with 1 to 2 R^(k) groups;

each R^(k) is independently selected from the group consisting of F, CN,methyl, ethyl and C₁₋₂ haloalkyl;

R¹ is

and

R² is selected from the group consisting of H, Cl, and F.

In some embodiments,

is selected from the group consisting of

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, the compound has the formula:

In some embodiments, the compound has the formula:

In some embodiments,

is selected from the group consisting of:

In some embodiments,

is selected from the group consisting of:

The disclosure also provides a compound, or a pharmaceuticallyacceptable salt thereof, of the formula (Ia) or (IIa):

wherein the variables are as defined in the embodiments above.

The disclosure also provides a compound, or a pharmaceuticallyacceptable salt thereof, of the formula (Ib) or (IIb):

wherein the variables are as defined in the embodiments above.

The disclosure also provides a compound, or a pharmaceuticallyacceptable salt thereof, of the formula (Ic) or (IIc):

wherein the variables are as defined in the embodiments above.

The disclosure also provides a compound, or a pharmaceuticallyacceptable salt thereof, of the formula (Id) or (IId):

wherein the variables are as defined in the embodiments above.

The disclosure also provides a compound, or a pharmaceuticallyacceptable salt thereof, of the formula (Ie) or (IIe):

wherein the variables are as defined in the embodiments above.

The disclosure also provides a compound, or a pharmaceuticallyacceptable salt thereof, of the formula (If), (IIf), (IIIf), (IVf),(Vf), (VIf), (VIIf) or (VIIIf):

wherein the variables are as defined in the embodiments above.

The invention also provides a compound, or a pharmaceutically acceptablesalt thereof, selected from the group consisting of

The invention also provides a compound, or a pharmaceutically acceptablesalt thereof, having formula (B):

or a pharmaceutically acceptable salt thereof, wherein

X¹ and X² are each independently selected from N and CH;

R^(d) and R^(e) are each independently selected from the groupconsisting of H and C₁₋₃ alkyl; optionally R^(d) and R^(e) may be joinedto form a cyclopropyl ring;

R^(k1) is selected from the group consisting of H, F, CN, OMe, and C₁₋₃alkyl;

R^(k2) is selected from the group consisting of H and methyl;

R¹ is

and

R² is selected from the group consisting of H, Cl, and F.

In some embodiments, R^(d) and R^(e) are each independently selectedfrom the group consisting of H and methyl; optionally R^(d) and R^(e)may be joined to form a cyclopropyl ring; and R^(k1) is selected fromthe group consisting of H, F, CN, OMe, methyl and ethyl.

The invention also provides a compound, or a pharmaceutically acceptablesalt thereof, having formula (C):

or a pharmaceutically acceptable salt thereof, wherein

X¹ and X² are both CH, or X¹ and X² are both N, or X¹ is N and X² is CH;

R^(d) and R^(e) are each independently selected from the groupconsisting of H and methyl;

R^(k) is selected from the group consisting of H, CN, methyl and ethyl;

R¹ is

and

R² is selected from the group consisting of H, Cl, and F.

In some embodiments, the compound or a pharmaceutically acceptable saltthereof, is selected from the group consisting of:

The invention also provides a compound, or a pharmaceutically acceptablesalt thereof, having the formula:

The invention also provides a compound, or a pharmaceutically acceptablesalt thereof, having the formula:

The invention also provides a compound, or a pharmaceutically acceptablesalt thereof, having the formula:

The invention also provides a pharmaceutical composition comprising acompound of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, and apharmaceutically-acceptable carrier. In some embodiments, thepharmaceutical composition further comprises one or more othertherapeutic agents. In some embodiments, the one or more othertherapeutic agent is useful for treating a gastrointestinal inflammatorydisease, an inflammatory disease of the skin, an inflammatory disease ofthe lungs or an inflammatory disease of the eye. In some embodiments,the one or more other therapeutic agent is useful for treating agastrointestinal inflammatory disease. In some embodiments thegastrointestinal inflammatory disease is ulcerative colitis. In someembodiments the gastrointestinal inflammatory disease is Crohn'sdisease.

Furthermore, some compounds may sometimes exist in tautomeric forms. Itwill be understood that although structures are shown, or named, in aparticular form, the invention also includes the tautomer thereof.

The compounds of the invention may contain one or more chiral centersand therefore, such compounds (and intermediates thereof) can exist asracemic mixtures; pure stereoisomers (i.e., enantiomers ordiastereomers); stereoisomer-enriched mixtures and the like. Chiralcompounds shown or named herein without a defined stereochemistry at achiral center are intended to include any or all possible stereoisomervariations at the undefined stereocenter unless otherwise indicated. Thedepiction or naming of a particular stereoisomer means the indicatedstereocenter has the designated stereochemistry with the understandingthat minor amounts of other stereoisomers may also be present unlessotherwise indicated, provided that the utility of the depicted or namedcompound is not eliminated by the presence of another stereoisomer.

This invention also includes isotopically-labeled compounds of thedisclosure, for example isotopically-labeled compounds of formula (I),(II), (B), (C), compound 1, compound 3, compound 4, i.e., compounds ofthe disclosure and compounds of formula (I), (II), (B), (C), compound 1,compound 3, compound 4 where an atom has been replaced or enriched withan atom having the same atomic number but an atomic mass different fromthe atomic mass that predominates in nature. Examples of isotopes thatmay be incorporated into a compounds of the disclosure and a compound offormula (I), (II), (B), (C), compound 1, compound 3, compound 4,include, but are not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³⁵S, and ¹⁸F. Of particular interest are compounds of thedisclosure and compounds of formula (I), (II), (B), (C), compound 1,compound 3, and compound 4, enriched in tritium or carbon-14, whichcompounds can be used, for example, in tissue distribution studies. Alsoof particular interest are compounds of the disclosure and compounds offormula (I), (II), (B), (C), compound 1, compound 3, compound 4 enrichedin deuterium especially at a site of metabolism, which compounds areexpected to have greater metabolic stability. Additionally, ofparticular interest are compounds of the disclosure and compounds offormula (I), (II), (B), (C), compound 1, compound 3, compound 4,enriched in a positron emitting isotope, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N,which compounds can be used, for example, in Positron EmissionTomography (PET) studies.

Definitions

When describing this invention including its various aspects andembodiments, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl (Me),ethyl (Et), n-propyl (n-Pr) or (nPr), isopropyl (i-Pr) or (iPr), n-butyl(n-Bu) or (nBu), sec-butyl, isobutyl, tert-butyl (t-Bu) or (tBu),n-pentyl, n-hexyl, 2,2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl,2-ethylbutyl, 2,2-dimethylpentyl, 2-propylpentyl, and the like.

The term “haloalkyl” refers to an alkyl group, as defined above, that issubstituted by one or more halogen, e.g., trifluoromethyl,difluoromethyl, trichloromethyl, 2,2,2 trifluoroethyl, 1,2difluoroethyl, 3 bromo 2 fluoropropyl, 1,2 dibromoethyl, and the like.

When a specific number of carbon atoms are intended for a particularterm, the number of carbon atoms is shown preceding the term. Forexample, the term “C₁₋₃ alkyl” means an alkyl group having from 1 to 3carbon atoms wherein the carbon atoms are in any chemically-acceptableconfiguration, including linear or branched configurations.

The term “alkoxy” means the monovalent group —O-alkyl, where alkyl isdefined as above. Representative alkoxy groups include, by way ofexample, methoxy, ethoxy, propoxy, butoxy, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclic groupwhich may be monocyclic or multicyclic. Unless otherwise defined, suchcycloalkyl groups typically contain from 3 to 10 carbon atoms.Representative cycloalkyl groups include, by way of example, cyclopropyl(cPr), cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,adamantyl, and the like.

The term “heterocycle”, “heterocyclic”, or “heterocyclic ring” means asaturated or partially unsaturated cyclic non-aromatic group, havingfrom 3 to 10 total ring atoms, wherein the ring contains from 2 to 9carbon ring atoms and from 1 to 4 ring heteroatoms selected fromnitrogen, oxygen, and sulfur. Heterocyclic groups may be monocyclic ormulticyclic (i.e., fused, spiro or bridged). When the heterocyclic groupis multicyclic, at least one but not necessarily all of the cyclicgroups contains a heteroatom. Representative heterocyclic groupsinclude, by way of example, pyrrolidinyl, piperidinyl, piperazinyl,imidazolidinyl, morpholinyl, thiomorpholyl, indolin-3-yl,2-imidazolinyl, tetrahydropyranyl, 1,2,3,4-tetrahydroisoquinolin-2-yl,quinuclidinyl, 7-azanorbornanyl, nortropanyl, and the like, where thepoint of attachment is at any available carbon or nitrogen ring atom.Where the context makes the point of attachment of the heterocyclicgroup evident, such groups may alternatively be referred to as anon-valent species, i.e. pyrrolidine, piperidine, piperazine, imidazole,tetrahydropyran etc.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition (such as a gastrointestinal inflammatorydisease), in a patient, such as a mammal (particularly a human) whichincludes one or more of the following:

(a) preventing the disease, disorder, or medical condition fromoccurring, i.e., preventing the reoccurrence of the disease or medicalcondition or prophylactic treatment of a patient that is pre-disposed tothe disease or medical condition;

(b) ameliorating the disease, disorder, or medical condition, i.e.,eliminating or causing regression of the disease, disorder, or medicalcondition in a patient, including counteracting the effects of othertherapeutic agents;

(c) suppressing the disease, disorder, or medical condition, i.e.,slowing or arresting the development of the disease, disorder, ormedical condition in a patient; or

(d) alleviating the symptoms of the disease, disorder, or medicalcondition in a patient.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts include saltsof acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. For example, the cation can be a protonated form ofa compound of formula (I), i.e. a form where one or more amino groupshave been protonated by an acid. Typically, the salt is apharmaceutically acceptable salt, although this is not required forsalts of intermediate compounds that are not intended for administrationto a patient.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl andtri-fluoroacetyl; alkoxycarbonyl groups, such as tert-butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS), triisopropylsiliyl (TIPS),tert-butyldimethylsilyl (TBS or TBDMS),[2-(trimethylsilyl)-ethoxy]methyl (SEM); and the like. Numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis,Third Edition, Wiley, New York

General Synthetic Procedures

Compounds of this disclosure, and intermediates thereof, can be preparedaccording to the following general methods and procedures usingcommercially-available or routinely-prepared starting materials andreagents. The substituents and variables (e.g., A, X¹, X², R¹, R², R³,R^(a), R^(b) etc.) used in the following schemes have the same meaningsas those defined elsewhere herein unless otherwise indicated.Additionally, compounds having an acidic or basic atom or functionalgroup may be used or may be produced as a salt unless otherwiseindicated (in some cases, the use of a salt in a particular reactionwill require conversion of the salt to a non-salt form, e.g., a freebase, using routine procedures before conducting the reaction).

Although a particular embodiment of the present invention may be shownor described in the following procedures, those skilled in the art willrecognize that other embodiments or aspects of the present invention canalso be prepared using such procedures or by using other methods,reagents, and starting materials known to those skilled in the art. Inparticular, it will be appreciated that compounds of the disclosure maybe prepared by a variety of process routes in which reactants arecombined in different orders to provide different intermediates en routeto producing final products.

A general method of preparing final compounds of the disclosure whereinL is selected from:

is illustrated in Scheme 1.

Starting material P1, where R^(x) and R^(y) are halogens which may bethe same or different, is protected with a protected group PG such astetrahydropyran to give P2. P2 is then reacted with P3 to give P4.

P3 may be:

where R^(z) is a second protecting group, for example Boc. In this case,P3 is deprotonated with a base such as NaH and reacted with P2 to giveP4.

Alternatively, P3 may be:

where R^(z) is a second protecting group, for example Boc. In this case,P3 is reacted with P2 under Buchwald coupling conditions such as in thepresence of Pd(0) and a base to give P4. Alternatively, P3 is reactedwith P2 in presence of a base such as DIPEA to give P4.

Alternatively, P3 may be:

where R^(z) is a second protecting group, for example Boc. In this case,P3 is reacted with P2 in presence of Pd(0), 9-BBN and a base to form P4.

P4 is coupled with boronic acid P5 (Suzuki coupling) in presence ofPd(0) and a base to give P6. P6 is deprotected to give P7 (when PG istetrahydropyran and R^(z) is Boc, simultaneous deprotection of theamines occur in presence of a strong acid such as TFA or HCl). Finally,P7 is derivatized into an amide by amide coupling (reaction with an acidin presence of a coupling agent such as HATU or hydroxybenzotriazole(HOBT)) or reaction with an acyl chloride in presence of a base such asHunig's base.

In this reaction scheme, the order of the reactions may be modified. Forexample the Suzuki coupling may be conducted before the introduction ofthe portion containing the A ring. This can for example be the case whenthe portion containing the A ring is introduced through a Buchwaldcoupling.

In this reaction scheme, protection of one or both of the amino groupsis optional. The same synthetic scheme may be used without aminoprotection for one or both amino groups but may provide lower yields.

The sulfonyl linker can be obtained by oxidizing the correspondingsulfide, for example with oxone and basic alumina.

Accordingly, in a method aspect, the invention provides a method forpreparing a compound of formula (I) or a pharmaceutically acceptablesalt thereof,

the method comprising:

reacting a compound of formula (III):

with

(i) or

(ii) HO— R¹

wherein R¹, R², R³, R⁴, X¹, X², L and A are as defined above, and

optionally forming a pharmaceutically-acceptable salt to provide acompound of formula (I), or a pharmaceutically acceptable salt thereof.

In separate and distinct aspects, the invention provides a compound offormula (III) wherein the variables take any of the values describedabove.

Crystalline Forms

In one aspect, the invention provides a crystalline form of the compoundof formula:

Form 1

Crystalline Form 1 of the invention is a crystalline anhydrous free formof compound 3. In one aspect, Form 1 is characterized by a powder X-raydiffraction (PXRD) pattern having significant diffraction peaks, amongother peaks, at 2θ values of 5.65±0.20, 14.22±0.20, 15.16±0.20, and19.31±0.20. Form 1 may be further characterized by a PXRD pattern havingadditional diffraction peaks at 2θ values of 7.12±0.20, 10.02±0.20,11.16±0.20, 17.06±0.20, and 24.43±0.20. Form 1 may be furthercharacterized by a PXRD pattern having two or more additionaldiffraction peaks, including three or more and four or more additionaldiffraction peaks at 20 values selected from 13.10±0.20, 14.82±0.20,16.55±0.20, 20.08±0.20, 21.08±0.20, 21.65±0.20, 22.51±0.20, 22.98±0.20,25.02±0.20, 25.72±0.20, 26.80±0.20, 27.06±0.20, 28.31±0.20, 30.08±0.20,30.31±0.20 and 32.08±0.20. Form 1 is characterized by a PXRD patternhaving three, four, five, or six diffraction peaks at 2θ values selectedfrom 5.65±0.20, 7.12±0.20, 10.02±0.20, 11.16±0.20, 14.22±0.20,15.16±0.20, 17.06±0.20, 19.31±0.20 and 24.43±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD pattern are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm 1 is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 1.

In another aspect, crystalline Form 1 is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 2, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits a peak in endothermic heat flow,identified as a melt transition, with an onset at about 154.9° C. and apeak at about 162.9° C. Melting was followed immediately bydecomposition.

The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow with a peak at about 162.9° C.The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow with a peak at 162.9±3° C.

The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow at a temperature between about154.9° C. and about 171° C., or between 158° C. and 167° C.

A representative TGA trace of the Form 1 crystalline free form is shownin FIG. 3. The thermal gravimetric analysis (TGA) trace shows a smallweight loss of about 0.14% at 100° C. The compound decomposes at anonset temperature of about 175° C.

A representative DMS trace for the Form 1 crystalline free form is shownin FIG. 4. Form 1 demonstrated about 1.62% weight gain in the humidityrange of 5% to 90% relative humidity. Form 1 is considered to beslightly hygroscopic.

Form 1 may be prepared by dissolving compound 3 as an amorphous form inethanol followed by stirring at a temperature of between about 20° C.and about 25° C. followed by filtration and drying to give Form 1.Optionally, the solid can be washed with ethanol before drying.

Form 1 may be prepared by adding acetone to the compound in an amorphousform and stirring at a temperature of between about 20° C. and about 25°C. followed by the addition of seeds. The resulting slurry is filteredand dried to give Form 1.

Form 2

Crystalline Form 2 of the invention is a crystalline hydrate free formof compound 3. In one aspect, Form 2 is characterized by a powder X-raydiffraction (PXRD) pattern having significant diffraction peaks, amongother peaks, at 2θ values of 6.90±0.20, 9.15±0.20, 10.00±0.20, and18.31±0.20. Form 2 may be further characterized by a PXRD pattern havingadditional diffraction peaks at 2θ values of 11.18±0.20, 15.51±0.20, and20.90±0.20. Form 2 may be further characterized by a PXRD pattern havingtwo or more additional diffraction peaks, including three or more andfour or more additional diffraction peaks at 2θ values selected from12.76±0.20, 13.33±0.20, 13.82±0.20, 14.43±0.20, 16.04±0.20, 17.00±0.20,17.90±0.20, 22.06±0.20, 22.51±0.20, 25.00±0.20, 26.92±0.20, 27.26±0.20,27.61±0.20, 29.37±0.20, 30.53±0.20, and 30.92±0.20. Form 2 ischaracterized by a PXRD pattern having three, four, five, or sixdiffraction peaks at 2θ values selected from 6.90±0.20, 9.15±0.20,10.00±0.20, 11.18±0.20, 15.51±0.20, 18.31±0.20, and 20.90±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD pattern are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm 2 is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 5.

In another aspect, crystalline Form 2 is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 6, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits a desolvation endotherm with an onset atabout 52.7° C. and a peak at about 84.4° C., and a melting endothermwith an onset at about 160.0° C. and a peak at about 167.6° C. Meltingwas followed immediately by decomposition.

A representative TGA trace of the Form 2 crystalline free form of theinvention is shown in FIG. 7. The thermal gravimetric analysis (TGA)trace of FIG. 7 shows a weight loss of about 6.73% at 75° C. Thecompound desolvates at an onset temperature of about 25° C. The compounddecomposes at an onset temperature of about 185° C.

A representative DMS trace for the Form 2 crystalline free form of theinvention is shown in FIG. 8. Form 2 converts to a hydrate (Form 2b) atRH above 65%. The dehydration occurs at RH below 15%. The total moistureuptake between 5%-90% RH is 7.99%.

Form 2 may be prepared by dissolving compound 3 in amorphous form inmethanol followed by the addition of an anti-solvent such as water, in aratio of about 1:2 methanol:water. Optionally, the mixture is sonicated.The mixture is then stirred at a temperature of between about 20° C. andabout 25° C. for about 12 to 24 hours. Form 2 is then isolated byfiltration and drying. Optionally, the solid can be washed withmethanol.

Form 2 can also be prepared by dissolution in ethanol and water ormethanol and water by complete dissolution in about 10 volumes ofalcohol followed by slow addition of about 8-10 volumes of water untilcloud point. Seeds of Form 2 are added to form a slurry slowly overtime. More water is added slowly (about 10 volumes) to provide a solidwhich can be filtered and dried to give Form 2.

Form 2b

Crystalline Form 2b of the invention is a crystalline dehydrated freeform of compound 3. In one aspect, Form 2b is characterized by a powderX-ray diffraction (PXRD) pattern having significant diffraction peaks,among other peaks, at 2θ values of 7.61±0.20, 16.76±0.20, 17.90±0.20,and 20.67±0.20. Form 2b may be further characterized by a PXRD patternhaving additional diffraction peaks at 2θ values of 10.33±0.20,11.25±0.20, 12.71±0.20, 15.88±0.20. Form 2b may be further characterizedby a PXRD pattern having two or more additional diffraction peaks,including three or more and four or more additional diffraction peaks at2θ values selected from 13.23±0.20, 13.66±0.20, 13.90±0.20, 15.02±0.20,15.27±0.20, 16.33±0.20, 18.26±0.20, 21.37±0.20, 21.92±0.20, 22.31±0.20,22.90±0.20, 23.22±0.20, 23.61±0.20, 24.74±0.20, 25.78±0.20, 26.23±0.20,26.73±0.20, 27.57±0.20, 29.10±0.20, 29.39±0.20, 30.72±0.20, 30.94±0.20,31.69±0.20, 32.06±0.20, 33.76±0.20, and 34.35±0.20. Form 2b ischaracterized by a PXRD pattern having three, four, five, or sixdiffraction peaks at 2θ values selected from 7.61±0.20, 10.33±0.20,11.25±0.20, 12.71±0.20, 15.88±0.20, 16.76±0.20, 17.90±0.20, and20.67±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD pattern are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm 2b is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 17.

In another aspect, the invention provides a crystalline form of thecompound of formula:

Form 3

Crystalline Form 3 of the invention is a crystalline anhydrous free formof compound 1. In one aspect, Form 3 is characterized by a powder X-raydiffraction (PXRD) pattern having significant diffraction peaks, amongother peaks, at 2θ values of 9.67±0.20, 11.61±0.20, 17.61±0.20,18.88±0.20, and 23.33±0.20. Form 3 may be further characterized by aPXRD pattern having additional diffraction peaks at 2θ values of4.82±0.20, 15.69±0.20, and 16.19±0.20. Form 3 may be furthercharacterized by a PXRD pattern having two or more additionaldiffraction peaks, including three or more and four or more additionaldiffraction peaks at 2θ values selected from 11.92±0.20, 12.98±0.20,13.23±0.20, 16.45±0.20, 16.67±0.20, 19.39±0.20, 19.96±0.20, 20.14±0.20,22.14±0.20, 23.84±0.20, 24.06±0.20, 24.29±0.20, 25.31±0.20, 25.63±0.20,27.06±0.20, 27.31±0.20, 30.10±0.20, and 30.53±0.20. Form 3 ischaracterized by a PXRD pattern having three, four, five, or sixdiffraction peaks at 2θ values selected from 4.82±0.20, 9.67±0.20,11.61±0.20, 15.69±0.20, 16.19±0.20, 17.61±0.20, 18.88±0.20, and23.33±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD pattern are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm 3 is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 9.

In another aspect, crystalline Form 3 is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 10, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits a peak in endothermic heat flow,identified as a melt transition, with an onset at about 197.7° C. and apeak at about 201.3° C. Melting was followed immediately bydecomposition.

The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow with a peak at 201.3° C.±2° C.

The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow at a temperature between 198°C. and 204° C.

A representative TGA trace of the Form 3 crystalline free form of theinvention is shown in FIG. 11. The thermal gravimetric analysis (TGA)trace of FIG. 11 shows no significant weight loss at temperatures belowthe onset of decomposition at about 195° C.

A representative DMS trace for the Form 3 crystalline free form of theinvention is shown in FIG. 12. Form 3 demonstrated about 0.33% weightgain in the humidity range of 5% to 90% relative humidity. Form 3 isconsidered to be non-hygroscopic.

Form 3 may be prepared by suspending compound 1 in an amorphous form ina 1:1 mixture of acetonitrile and isopropanol. The resulting suspensionis stirred for about 1 day at about 50° C., filtered, optionally washedwith a 1:1 mixture of acetonitrile and isopropanol and dried for a fewhours to provide Form 3.

Form 3 may be prepared by dissolving compound 1 as an amorphous freebase in IPA at a temperature of between about 20° C. and about 25° C. Anequal amount of acetonitrile is added. More compound can be added untila saturated solution is formed. Seeds are added and the mixture isstirred overnight. The developing white slurry formed is filtered anddried to yield Form 3.

Form 4

Crystalline Form 4 of the invention is a crystalline hydrate free formof compound 1. In one aspect, Form 4 is characterized by a powder X-raydiffraction (PXRD) pattern having significant diffraction peaks, amongother peaks, at 2θ values of 6.26±0.20, 16.55±0.20, 16.94±0.20,18.33±0.20, 23.61±0.20, and 24.24±0.20. Form 4 may be furthercharacterized by a PXRD pattern having additional diffraction peaks at20 values of 11.86±0.20, 12.51±0.20, 13.16±0.20, and 14.98±0.20. Form 4may be further characterized by a PXRD pattern having two or moreadditional diffraction peaks, including three or more and four or moreadditional diffraction peaks at 20 values selected from 17.61±0.20,18.78±0.20, 19.39±0.20, 19.57±0.20, 19.84±0.20, 21.45±0.20, 21.82±0.20,22.57±0.20, 24.67±0.20, 25.10±0.20, 25.39±0.20, 27.19±0.20, 27.39±0.20,28.55±0.20, and 31.51±0.20. Form 4 is characterized by a PXRD patternhaving three, four, five, or six diffraction peaks at 2θ values selectedfrom 6.26±0.20, 11.86±0.20, 12.51±0.20, 13.16±0.20, 14.98±0.20,16.55±0.20, 16.94±0.20, 18.33±0.20, 23.61±0.20, and 24.24±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD pattern are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm 4 is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 13.

In another aspect, crystalline Form 4 is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 14, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits a desolvation endotherm with an onset atabout 60.9° C. and a peak at about 103.6° C., and a melting endothermcharacterized by an onset at about 167.3° C. The compound decomposes atmelting and the melting endotherm and the decomposition exothermoverlap.

A representative TGA trace of the Form 4 crystalline free form of theinvention is shown in FIG. 15. The thermal gravimetric analysis (TGA)trace of FIG. 15 shows a weight loss of about 3.54% at 100° C. Thecompound desolvates at an onset temperature of about 50° C. The compounddecomposes at an onset temperature of about 165° C.

A representative DMS trace for the Form 4 crystalline free form of theinvention is shown in FIG. 16. Form 4 demonstrated about 5.01% weightgain in the humidity range of 5% to 90% relative humidity. Form 4 isconsidered to be moderately hygroscopic.

Form 4 may be prepared by suspending compound 1 in water. The resultingsuspension is stirred for about 1 to 2 days at about 50° C., filtered,optionally washed with water, and dried at a temperature of betweenabout 20° C. and about 25° C. for about 2-6 hours to provide Form 4.

Alternatively, Form 4 may be prepared by dissolving compound 1 inethanol and water or methanol and water by complete dissolution in about10 volumes of alcohol followed by slow addition of about 8-10 volumes ofwater until cloud point. Seeds of Form 4 are added and a resultingslurry develops slowly over time. Then more water is added slowly (about10 volumes) and the solid is filtered and dried to give Form 4.

Pharmaceutical Compositions

The compounds of the disclosure and pharmaceutically-acceptable saltsthereof are typically used in the form of a pharmaceutical compositionor formulation. Such pharmaceutical compositions may be administered toa patient by any acceptable route of administration including, but notlimited to, oral, topical (including transdermal), rectal, nasal,inhaled, and parenteral modes of administration.

Accordingly, in one of its composition aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a compound offormula (I), (II), (B), (C), compound 1, compound 3, or compound 4 or apharmaceutically-acceptable salt thereof. Optionally, suchpharmaceutical compositions may contain other therapeutic and/orformulating agents if desired. When discussing compositions and usesthereof, the “compound of the invention” or “compound of the disclosure”may also be referred to herein as the “active agent”. As used herein,the term “compound(s) of the disclosure” is intended to include allcompounds encompassed by formula (I), (II), (B), (C), (Ia), (IIa), (Ib),(IIb), (Ic), (IIc), (Id), (IId), (Ie), (IIe), (If), (IIf), (IVf), (Vf),(VIf), (VIIf), and (VIIIf), and pharmaceutically-acceptable saltsthereof.

The pharmaceutical compositions of the disclosure typically contain atherapeutically effective amount of a compound of the disclosure. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.1to about 95% by weight of the active agent; including from about 5 toabout 70% by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the disclosure are preferablypackaged in a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor parenteral administration.

In one embodiment, the pharmaceutical compositions of the disclosure aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of a compound of the presentdisclosure as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the disclosure will typically comprise the active agent and one ormore pharmaceutically-acceptable carriers. Optionally, such solid dosageforms may comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, dicalcium phosphate, sucrose,glucose, mannitol, and/or silicic acid; binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as crosscarmellose sodium, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the disclosure. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid, methacrylic acid estercopolymers, cellulose acetate trimellitate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, and thelike.

Pharmaceutical compositions of the disclosure may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methylcellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the disclosure may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), oleic acid, glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Alternatively, certain liquid formulations can be converted,for example, by spray drying, to a powder, which is used to preparesolid dosage forms by conventional procedures.

Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

The compounds of this disclosure, or a pharmaceutically acceptable saltthereof, can also be administered parenterally (e.g. by intravenous,subcutaneous, intramuscular or intraperitoneal injection). Forparenteral administration, the active agent is typically admixed with asuitable vehicle for parenteral administration including, by way ofexample, sterile aqueous solutions, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, vegetable oils,gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,buffering agents, or dispersing agents. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat.

Alternatively, the pharmaceutical compositions of the disclosure areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the disclosure will typically comprisethe active ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the disclosure and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The compounds of the disclosure, or a pharmaceutically acceptable saltthereof, may also be formulated for topical administration to the skinas an ointment or cream. Ointment formulations are semisolidpreparations having a base of an oily or greasy material that istypically clear. Suitable oily materials for use in ointmentformulations include petrolatum (petroleum jelly), beeswax, cocoabutter, shea butter, and cetyl alcohol. Ointments may optionallyadditionally include emollients and penetration enhancers, if desired.

Cream formulations may be prepared as emulsions comprising an oil phaseand aqueous phase, typically including purified water. Components ofcream formulations may include: oil bases, such as petrolatrum, mineraloils, vegetable and animal oils, and triglycerides; cream bases, such aslanolin alcohols, stearic acid, and cetostearyl alcohol; a gel base,such as polyvinyl alcohol; solvents, such as, propylene glycol andpolyethylene glycol; emulsifiers, such as polysorbates, stearates, suchas glyceryl stearate, octylhydroxystearate, polyoxyl stearate, PEGstearyl ethers, isopropyl palmitate, and sorbitan monostearate;stabilizers, such as polysaccharides and sodium sulfite; emollients(i.e.moisturizers), such as medium chain triglycerides, isopropylmyristate, and dimethicone; stiffening agents, such as cetyl alcohol andstearyl alcohol; antimicrobial agents, such as methylparaben,propylparaben, phenoxyethanol, sorbic acid, diazolidinyl urea, andbutylated hydroxyanisole; penetration enhancers, such asN-methylpyrrolidone, propylene glycol, polyethylene glycol monolaurate,and the like; and chelating agents, such as edetate disodium.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Tablet Oral Solid Dosage Form

A compound of the disclosure or a pharmaceutically-acceptable saltthereof is dry blended with microcrystalline cellulose, polyvinylpyrrolidone, and crosscarmellose sodium in a ratio of 4:5:1:1 andcompressed into tablets to provide a unit dosage of, for example, 5 mg,20 mg or 40 mg active agent per tablet.

Capsule Oral Solid Dosage Form

A compound of the disclosure or a pharmaceutically-acceptable saltthereof is combined with microcrystalline cellulose, polyvinylpyrrolidone, and crosscarmellose sodium in a ratio of 4:5:1:1 by wetgranulation and loaded into gelatin or hydroxypropyl methylcellulosecapsules to provide a unit dosage of, for example, 5 mg, 20 mg or 40 mgactive agent per capsule.

Liquid Formulation A liquid formulation comprising a compound of thedisclosure (0.1%), water (98.9%) and ascorbic acid (1.0%) is formed byadding a compound of the disclosure to a mixture of water and ascorbicacid.

Enteric Coated Oral Dosage Form

A compound of the disclosure is dissolved in an aqueous solutioncontaining polyvinyl pyrrolidone and spray coated onto microcrystallinecellulose or sugar beads in a ratio of 1:5 w/w active agent:beads andthen an approximately 5% weight gain of an enteric coating comprising anacrylic copolymer, for example a combination of acrylic copolymersavailable under the trade names Eudragit-L® and Eudragit-S®, orhydroxypropyl methylcellulose acetate succinate is applied. The entericcoated beads are loaded into gelatin or hydroxypropyl methylcellulosecapsules to provide a unit dosage of, for example, 30 mg active agentper capsule.

Enteric Coated Oral Dosage Form

An enteric coating comprising a combination of Eudragit-L® andEudragit-S®, or hydroxypropyl methylcellulose acetate succinate isapplied to a tablet oral dosage form or a capsule oral dosage formdescribed above.

Ointment Formulation for Topical Administration

A compound of the disclosure is combined with petrolatum, C₈-C₁₀triglyceride, octylhydroxystearate, and N-methylpyrrolidone in a ratioto provide a composition containing 0.05% to 5% active agent by weight.

Ointment Formulation for Topical Administration

A compound of the disclosure is combined with white petrolatum,propylene glycol, mono- and di-glycerides, paraffin, butylatedhydroxytoluene, and edetate calcium disodium in a ratio to provide acomposition containing 0.05% to 5% active agent by weight.

Ointment Formulation for Topical Administration

A compound of the disclosure is combined with mineral oil, paraffin,propylene carbonate, white petrolatum and white wax to provide acomposition containing 0.05% to 5% active agent by weight.

Cream Formulation for Topical Administration Mineral oil is combinedwith a compound of the disclosure, propylene glycol, isopropylpalmitate, polysorbate 60, cetyl alcohol, sorbitan monostearate,polyoxyl 40 stearate, sorbic acid, methylparaben and propylparaben toform an oil phase, which is combined with purified water by shearblending to provide a composition containing 0.05% to 5% active agent byweight.

Cream Formulation for Topical Administration

A cream formulation comprising a compound of the disclosure, benzylalcohol, cetyl alcohol, citric acid anhydrous, mono and di-glycerides,oleyl alcohol, propylene glycol, sodium cetostearyl sulphate, sodiumhydroxide, stearyl alcohol, triglycerides, and water contains 0.05% to5% active agent by weight.

Cream Formulation for Topical Administration

A cream formulation comprising a compound of the disclosure, cetostearylalcohol, isopropyl myristate, propylene glycol, cetomacrogol 1000,dimethicone 360, citric acid, sodium citrate, and purified water, withimidurea, methylparaben, and propylparaben, as preservatives, contains0.05% to 5% active agent by weight.

Utility

Inhibition of JAK3 blocks the signaling of many key pro-inflammatorycytokines. Thus the compounds of the disclosure are expected to beuseful in the treatment of inflammatory diseases.

The compounds of the disclosure have been designed to be selective forJAK3 over JAK1, JAK2 and TYK2. Selectivity for JAK3 over JAK1 isanticipated to be beneficial as there is some evidence that JAK3selectivity allows sparing of potentially beneficial cytokines such asIL-10 which has been involved in mucosal healing, IL-22 which isinvolved in mucus barrier protection and epithelial regeneration, andIL-6 which is involved in the proliferation of intestinal epithelialcells. Selectivity for JAK3 over JAK2 allows sparing of erythropoietin(EPO) and thrombopoietin (TPO) signaling.

Without being limited by this theory, the compounds of the disclosurepossess an electrophilic portion which may form a covalent bond with thecysteine (Cys909) present in JAK3, a residue replaced by a serine in theother three JAK isoforms (Goedken et al., J Biol Chem., 2015, 290, 8,4573-89). Such covalent binding to JAK3 could be beneficial by providingan extended target engagement which may translate in better efficacy. Asdescribed in the experimental part, co-crystal structures of compounds1, 3 and 4 covalently bound to human JAK3 have been obtained whichconfirms the irreversible binding nature for each of these ligands toJAK3.

Some compounds of the disclosure have been designed to have their effectat the site of action without significant systemic effects, therebyavoiding the potential adverse systemic immunosuppressive effects.

Gastrointestinal Inflammatory Disease

In addition to providing potent inhibition of JAK3, some compounds ofthe disclosure have been designed to be poorly absorbed to minimizesystemic exposure. These compounds are designed to have their effect atthe site of action, for example, in the colon. As described in Assay 6,certain compounds exhibit low permeabilities with K_(p) values less thanabout 5×10⁻⁶ cm/sec which is considered favorable to minimize systemicexposure and target the colon. Certain compounds have a K_(p) value lessthan about 10×10⁻⁶ cm/sec which may also be sufficient to minimizesystemic exposure and target the colon. As described in Assay 7 below,compounds 1, 2, 3, 4, 6, 7, 8, 21 and 22 exhibited a ratio of exposurein the colon to exposure in plasma upon oral administration greater thanabout 1250. Compounds 9, 5, 19, and 20 exhibited a colon to plasma ratioin excess of about 200.

Oxazolone-induced colitis is an experimental model that has ahistological resemblance to human ulcerative colitis. As described inAssay 8, the compounds 1, 2, 3, 4, 5, 6, 7, 8, 3-11, 5-10, 19, 15-1,3-55, 3-34, 15-3, 21, 3-80, 3-81, 3-72, 3-57, 3-113 and 3-74demonstrated activity in the oxazolone-induced colitis model in mice.Further, when tested in Assay 9, an immunosuppression model in mice,which probes systemic functional activity, splenic NK cell counts wereunaffected by compounds 1, 2, 4, 5, and 8 at the same or higher dosesrequired to demonstrate efficacy in the oxazolone model.

Finally, compounds 1, 2, 3, 4, 5, 6, 7, and 8 were shown to demonstratelack of systemic activity in a murine model of IL-2 induced pSTAT5induction in the thymus.

Thus these compounds demonstrated anti-colitic activity withoutexhibiting systemic effects in preclinical models.

It is expected that a high colon to plasma ratio will provide robust,luminally-driven anti-inflammatory activity without associated,systemically-driven, adverse effects. Such compounds may be useful for avariety of gastrointestinal inflammatory indications that include, butare not limited to, inflammatory bowel disease, ulcerative colitis(proctosigmoiditis, pancolitis, ulcerative proctitis and left-sidedcolitis), Crohn's disease, collagenous colitis, lymphocytic colitis,Behcet's disease, celiac disease, immune checkpoint inhibitor inducedcolitis, ileitis, eosinophilic esophagitis, graft versus hostdisease-related colitis, and infectious colitis. Ulcerative colitis(Reimund et al., J Clin Immunology, 1996, 16, 144-150), Crohn's disease(Woywodt et al., Eur J Gastroenterology Hepatology, 1999, 11, 267-276),collagenous colitis (Kumawat et al., Mol Immunology, 2013, 55, 355-364),lymphocytic colitis (Kumawat et al., 2013), eosinophilic esophagitis(Weinbrand-Goichberg et al., Immunol Res, 2013, 56, 249-260), graftversus host disease-related colitis (Coghill et al., Blood, 2001, 117,3268-3276), infectious colitis (Stallmach et al., Int J Colorectal Dis,2004, 19, 308-315), Behcet's disease (Zhou et al., Autoimmun Rev, 2012,11, 699-704), celiac disease (de Nitto et al., World J Gastroenterol,2009, 15, 4609-4614), immune checkpoint inhibitor induced colitis (e.g.,CTLA-4 inhibitor-induced colitis; (Yano et al., J Translation Med, 2014,12, 191), PD-1- or PD-L1-inhibitor-induced colitis), and ileitis(Yamamoto et al., Dig Liver Dis, 2008, 40, 253-259) are characterized byelevation of certain pro-inflammatory cytokine levels. As manypro-inflammatory cytokines signal via JAK activation, compoundsdescribed in this application may be able to alleviate the inflammationand provide symptom relief

In particular, the compounds of the disclosure may be useful for theinduction and maintenance of remission of ulcerative colitis, and forthe treatment of Crohn's disease, immune checkpoint inhibitor inducedcolitis, and the gastrointestinal adverse effects in graft versus hostdisease.

In one aspect, therefore, the invention provides a method of treating agastrointestinal inflammatory disease in a mammal (e.g., a human), themethod comprising administering to the mammal a compound of thedisclosure, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of the disclosure, or a pharmaceuticallyacceptable salt thereof.

The invention further provides a method of treating ulcerative colitisin a mammal, the method comprising administering to the mammal acompound of the disclosure, or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of the disclosure, ora pharmaceutically acceptable salt thereof.

When used to treat ulcerative colitis, the compounds of the disclosure,or a pharmaceutically acceptable salt thereof will typically beadministered orally in a single daily dose or in multiple doses per day,although other forms of administration may be used. The amount of activeagent administered per dose or the total amount administered per daywill typically be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like. Suitabledoses for treating ulcerative colitis and other gastrointestinalinflammatory disorders are expected to range from about 1 to about 400mg/day of active agent, including from about 5 to about 300 mg/day andfrom about 20 to about 70 mg per day of active agent for an average 70kg human.

Combination therapy Compounds of the disclosure or a pharmaceuticallyacceptable salt thereof may also be used in combination with one or moreagents which act by the same mechanism or by different mechanisms toeffect treatment of gastrointestinal inflammatory disorders. Thedifferent agents may be administered sequentially or simultaneously (inseparate compositions or in the same composition). Useful classes ofagents for combination therapy include, but are not limited to,aminosalicylates, steroids, systemic immunosuppressants, anti-TNFαantibodies, TNF alpha ligand inhibitor, TNF binding agent, anti-VLA-4antibodies, anti-integrin α₄β₇ antibodies, anti-bacterial agents,Glucocorticoid agonists, Nuclear factor kappa B inhibitors,5-Lipoxygenase inhibitors, integrin alpha-4/beta-7 antagonist,Cyclooxygenase inhibitors, IL-23 antagonists, Leukotriene BLT receptorantagonist, IL-6 antagonists, IL-8 antagonists, integrin antagonists,nicotinic acetylcholine receptor agonists, PPAR gamma agonists,sphingosine-1-phosphate receptor-1 modulators, B-lymphocyte antigen CD20inhibitors, calcineurin inhibitors, CD3 antagonist, cell adhesionmolecule inhibitors, eosinophil peroxidase inhibitors, heparin agonists,ICAM1 gene inhibitors, IL-13 antagonists, IL-2 receptor alpha subunitinhibitors, insulin sensitizers, interferon beta ligands, interferongamma receptor antagonists, interleukin-1 beta ligand modulators, MAdCAMinhibitors, PDE 4 inhibitors, sphingosine-1-phosphate receptor-1agonists, TLR-9 agonists, acetylcholinesterase inhibitors, ACTH receptoragonists, activin receptor antagonists, CCR5 chemokine antagonists, CCR9chemokine antagonists, and anti-diarrheal medicines.

Aminosalicylates that may be used in combination with the present JAKinhibitor compounds include, but are not limited to, mesalamine,osalazine and sulfasalazine. Examples of steroids include, but are notlimited to, prednisone, prednisolone, hydrocortisone, budesonide,beclomethasone, and fluticasone. Systemic immunosuppressants useful fortreatment of inflammatory disorders include, but are not limited tocyclosporine, azathioprine, methotrexate, 6-mercaptopurine, andtacrolimus. Further, anti-TNFα antibodies, which include, but are notlimited to, infliximab, adalimumab, golimumab, and certolizumab, may beused in combination therapy. Useful compounds acting by other mechanismsinclude anti-VLA-4 antibodies, such as natalizumab, anti-integrin α₄β₇antibodies, such as vedolizumab, anti-bacterial agents, such asrifaximin, and anti-diarrheal medicines, such as loperamide. (Mozaffariet al. Expert Opin. Biol. Ther.2014, 14, 583-600; Danese, Gut, 2012, 61,918-932; Lam et al., Immunotherapy, 2014, 6, 963-971.)

Other compounds that may be used in combination with the present JAKinhibitor compounds include, but are not limited to opaganib, abatacept,mongersen, filgotinib, LYC-30937, BI-655130, mirikizumab, adalimumab,tacrolimus, rituximab, GSK-2982772, andecaliximab, naltrexone,risankizumab, QBECO, alicaforsen, etrolizumab, foralumab, ocrelizumab,vedolizumab, amiselimod, ozanimod, dolcanatide, catridecacog,budesonide, STNM-01, cannabidiol, telotristat etiprate, SHP-647,carotegrast methyl, peg-ilodecakin, TOP-1288, iberogast N, PF-06480605,peficitinib, beclomethasone, recombinant interferon beta-1a, infliximab,golimumab, tralokinumab, ustekinumab, certolizumab pegol, thalidomide,upadacitinib, apremilast, natalizumab, interferon beta-1a, rifaximin,RBX-2660, etrasimod, zileuton, fingolimod, cobitolimod, ropivacaine,ABX-464, PF-06700841, prednisolone, GLPG-0974, valganciclovir,ciclosporin, VB-201, tulinercept, MDGN-002, PTG-100, dexamethasone,GED-0507-34-Levo, bertilimumab, brazikumab, KHK-4083, rosiglitazone,mocravimod, sotrastaurin, KAG-308, PUR-0110, E-6007, balsalazide,basiliximab, LP-02, ASP-3291, Trichuris suis ova, K(D)PT, midismase,DNVX-078, vatelizumab, alequel, low molecular weight heparin,metenkefalin, tridecactide, HMPL-004, SB-012, olsalazine, balsalazide,propionyl-L-carnitine, Clostridium butyricum, beclomethasone andacemannan.

In another aspect, therefore, the invention provides a therapeuticcombination for use in the treatment of gastrointestinal inflammatorydisorders, the combination comprising a compound of the disclosure, or apharmaceutically acceptable salt thereof, and one or more othertherapeutic agents useful for treating gastrointestinal inflammatorydisorders, such as the ones illustrated above. For example, theinvention provides a combination comprising a compound of thedisclosure, or a pharmaceutically acceptable salt thereof, and one ormore agents selected from aminosalicylates, steroids, systemicimmunosuppressants, anti-TNFα antibodies, anti-VLA-4 antibodies,anti-integrin α₄β₇ antibodies, anti-bacterial agents, and anti-diarrhealmedicines. Secondary agent(s), when included, are present in atherapeutically effective amount, i.e. in any amount that produces atherapeutically beneficial effect when co-administered with a compoundof the disclosure or a pharmaceutically acceptable salt thereof. Alsoprovided, therefore, is a pharmaceutical composition comprising acompound of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, and one or more othertherapeutic agents useful for treating gastrointestinal inflammatorydisorders.

Further, in a method aspect, the invention provides a method of treatinggastrointestinal inflammatory disorders, the method comprisingadministering to the mammal a compound of the disclosure, or apharmaceutically acceptable salt thereof, or a crystalline form thereof,and one or more other therapeutic agents useful for treatinggastrointestinal inflammatory disorders.

When used in combination therapy, the agents may be formulated in asingle pharmaceutical composition, as disclosed above, or the agents maybe provided in separate compositions that are administeredsimultaneously or at separate times, by the same or by different routesof administration. When administered separately, the agents areadministered sufficiently close in time so as to provide a desiredtherapeutic effect. Such compositions can be packaged separately or maybe packaged together as a kit. The two or more therapeutic agents in thekit may be administered by the same route of administration or bydifferent routes of administration.

Inflammatory skin disease Atopic dermatitis and other inflammatory skindiseases have been associated with elevation of proinflammatorycytokines that rely on the JAK-STAT pathway. Therefore, the compounds ofthe disclosure, or a pharmaceutically acceptable salt thereof, or acrystalline form thereof, may be beneficial in a number of dermalinflammatory or pruritic conditions that include, but are not limited toatopic dermatitis, alopecia areata, vitiligo, psoriasis,dermatomyositis, cutaneous T cell lymphoma (Netchiporouk et al., CellCycle. 2014; 13, 3331-3335) and subtypes (Sezary syndrome, mycosisfungoides, pagetoid reticulosis, granulomatous slack skin, lymphomatoidpapulosis, pityriasis lichenoides chronica, pityriasis lichenoides etvarioliformis acuta, CD30+ cutaneous T-cell lymphoma, secondarycutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30−cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennertlymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blasticNK-cell lymphoma), prurigo nodularis, lichen planus, primary localizedcutaneous amyloidosis, bullous pemphigoid, skin manifestations of graftversus host disease, pemphigoid, discoid lupus, granuloma annulare,lichen simplex chronicus, vulvar/scrotal/perianal pruritus, lichensclerosus, post herpetic neuralgia itch, lichen planopilaris, andfoliculitis decalvans. In particular, atopic dermatitis (Bao et al.,JAK-STAT, 2013, 2, e24137), alopecia areata (Xing et al., Nat Med. 2014,20, 1043-1049), vitiligo (Craiglow et al, JAMA Dermatol. 2015, 151,1110-1112), prurigo nodularis (Sonkoly et al., J Allergy Clin Immunol.2006, 117, 411-417), lichen planus (Welz-Kubiak et al., J Immunol Res.2015, ID:854747), primary localized cutaneous amyloidosis (Tanaka etal., Br J Dermatol. 2009, 161, 1217-1224), bullous pemphigoid (Felicianiet al., Int J Immunopathol Pharmacol. 1999, 12, 55-61), and dermalmanifestations of graft versus host disease (Okiyama et al., J InvestDermatol. 2014, 134, 992-1000) are characterized by elevation of certaincytokines that signal via JAK activation. Accordingly, compounds of thedisclosure, or a pharmaceutically acceptable salt thereof, or acrystalline form thereof, may be able to alleviate associated dermalinflammation or pruritus driven by these cytokines. In particular,compounds of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, may be expected to be useful forthe treatment of atopic dermatitis and other inflammatory skin diseases.

In one aspect, therefore, the invention provides a method of treating aninflammatory skin disease in a mammal (e.g., a human), the methodcomprising applying a pharmaceutical composition comprising a compoundof the disclosure, or a pharmaceutically acceptable salt thereof, or acrystalline form thereof, and a pharmaceutical carrier to the skin ofthe mammal. In one aspect, the inflammatory skin disease is atopicdermatitis.

Compounds of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, may also be used in combinationwith one or more compound useful to treat inflammatory skin diseases. Insome embodiments, the one or more compound is a steroid, Histamine H1receptor antagonist, calcineurin inhibitor, IL-13 antagonist, PDE 4inhibitor, G-protein coupled receptor-44 antagonist, IL-4 antagonist,5-HT 1a receptor antagonist, 5-HT 2b receptor antagonist, Alpha 2adrenoceptor agonist, cannabinoid CB1 receptor antagonist, CCR3chemokine, antagonist, collagenase inhibitor, cytosolic phospholipase A2inhibitor, eotaxin ligand inhibitor, GATA 3 transcription factorinhibitor, Histamine H4 receptor antagonist, IL-10 antagonist, IL-12antagonist, IL-17 antagonist, IL-2 antagonist, IL-23 antagonist, IL-4receptor modulator, IL-5 antagonist, immunoglobulin E antagonist,immunoglobulin E modulator, interferon gamma receptor antagonist,Interleukin 33 ligand inhibitor, Interleukin-31 receptor antagonist,Leukotriene antagonist, Liver X receptor agonist, Liver X receptor betaagonist, nuclear factor kappa B inhibitor, OX-40 receptor antagonist,PGD2 antagonist, phospholipase A2 inhibitor, SH2 domain inositolphosphatase 1 stimulator, thymic stromal lymphoprotein ligand inhibitor,TLR modulator, TNF alpha ligand modulator, or vanilloid VR1 antagonist.In some embodiments, the one or more compound is a gram positiveantibiotic, such as mupirocin or fusidic acid. In some embodiments, theone or more compound is tranilast, tacrolimus, epinastine, SB-011,AM-1030, ZPL-521, MM-36, FB-825, PG-102, viromed, GBR-830, AVX-001,AMG-0101, E-6005, DMT-210, AX-1602, bertilimumab, rosiptor acetate,Q-301, ANB-020, VTP-38543, ZPL-389, lebrikizumab, tezepelumab,fexofenadine, pimecrolimus, bepotastine, crisaborole, tralokinumab,fevipiprant, doxycycline, desloratadine, ALX-101, nemolizumab,asivatrep, ciclosporin, mepolizumab, dupilumab, secukinumab,timapiprant, or ustekinumab.

In one aspect, therefore, the invention provides a method of treating aninflammatory skin disease in a mammal, the method comprising applying acompound of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, and a gram positive antibioticto the skin of the mammal. In another aspect, the invention provides apharmaceutical composition comprising a compound of the disclosure, or apharmaceutically acceptable salt thereof, or a crystalline form thereof,a gram positive antibiotic, and a pharmaceutically-acceptable carrier.

Respiratory Diseases

Cytokines which signal through the JAK-STAT pathway, in particular IL-2,IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13, IL-23, IL-31, IL-27, thymicstromal lymphopoietin (TSLP), interferon-γ (IFNγ) andgranulocyte-macrophage colony-stimulating factor (GM-CSF) have also beenimplicated in asthma inflammation and in other inflammatory respiratorydiseases. As described above, the compounds of the disclosure have beenshown to be potent inhibitors of JAK3 and have also demonstrated potentinhibition of IL-2 pro-inflammatory cytokines in cellular assays.

The anti-inflammatory activity of JAK inhibitors has been robustlydemonstrated in preclinical models of asthma (Malaviya et al., IntImmunopharmacol, 2010, 10, 829,-836; Matsunaga et al., Biochem andBiophys Res Commun, 2011, 404, 261-267; Kudlacz et al., Eur J Pharmacol,2008, 582, 154-161.) Accordingly, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, or a crystalline form thereof,may be useful for the treatment of inflammatory respiratory disorderssuch as asthma. Inflammation and fibrosis of the lung is characteristicof other respiratory diseases in addition to asthma such as chronicobstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, and bronchiolitis obliterans. The compounds of thedisclosure, or a pharmaceutically acceptable salt thereof, therefore,may be useful for the treatment of chronic obstructive pulmonarydisease, cystic fibrosis, pneumonitis, interstitial lung diseases(including idiopathic pulmonary fibrosis), acute lung injury, acuterespiratory distress syndrome, bronchitis, emphysema, bronchiolitisobliterans, bronchiolitis obliterans organizing pneumonia (also termedCOS), primary Graft Dysfunction (PGD), organizing pneumonia (OP), acuterejection (AR), lymphocytic bronchiolitis (LB), chronic Lung AllograftDysfunction (CLAD), restrictive CLAD (rCLAD or RAS), neutrophilicallograft dysfunction, and sarcoidosis.

In one aspect, therefore, the disclosure provides a method of treating arespiratory disease in a mammal (e.g., a human), the method comprisingadministering to the mammal a compound of the disclosure or apharmaceutically-acceptable salt thereof, or a crystalline form thereof.

In one aspect, the respiratory disease is asthma, chronic obstructivepulmonary disease, cystic fibrosis, pneumonitis, chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, bronchiolitis obliterans, bronchiolitis obliterans organizingpneumonia (also termed COS), primary Graft Dysfunction (PGD), organizingpneumonia (OP), acute rejection (AR), lymphocytic bronchiolitis (LB),chronic Lung Allograft Dysfunction (CLAD), restrictive CLAD (rCLAD orRAS), neutrophilic allograft dysfunction, allergic rhinitis orsarcoidosis. In another aspect, the respiratory disease is asthma orchronic obstructive pulmonary disease.

In a further aspect, the respiratory disease is a lung infection, ahelminthic infection, pulmonary arterial hypertension, sarcoidosis,lymphangioleiomyomatosis, bronchiectasis, or an infiltrative pulmonarydisease. In yet another aspect, the respiratory disease is drug-inducedpneumonitis, fungal induced pneumonitis, allergic bronchopulmonaryaspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosiswith polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathicchronic eosinophilic pneumonia, hypereosinophilic syndrome, Lofflersyndrome, bronchiolitis obliterans organizing pneumonia, orimmune-checkpoint-inhibitor induced pneumonitis.

The disclosure further provides a method of treating a respiratorydisease, the method comprising administering to the mammal apharmaceutical composition comprising a compound of the disclosure or apharmaceutically-acceptable salt thereof, or a crystalline form thereof,and a pharmaceutically-acceptable carrier.

Compounds of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, may also be used in combinationwith one or more compound useful to respiratory diseases.

Ocular Diseases

Many ocular diseases have been shown to be associated with elevations ofproinflammatory cytokines that rely on the JAK-STAT pathway.

The compounds of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, therefore, may be useful for thetreatment of a number of ocular diseases that include, but are notlimited to, uveitis, diabetic retinopathy, diabetic macular edema, dryeye disease, age-related macular degeneration, retinal vein occlusion(RVO) and atopic keratoconjunctivitis.

In particular, uveitis (Horai and Caspi, J Interferon Cytokine Res,2011, 31, 733-744), diabetic retinopathy (Abcouwer, J Clin Cell Immunol,2013, Suppl 1, 1-12), diabetic macular edema (Sohn et al., AmericanJournal of Opthamology, 2011, 152, 686-694), dry eye disease (Stevensonet al, Arch Ophthalmol, 2012, 130, 90-100), retinal vein occlusion(Shchuko et al, Indian Journal of Ophthalmology, 2015, 63(12), 905-911)and age-related macular degeneration (Knickelbein et al, Int OphthalmolClin, 2015, 55(3), 63-78) are characterized by elevation of certainpro-inflammatory cytokines that signal via the JAK-STAT pathway.Accordingly, compounds of the disclosure, or a pharmaceuticallyacceptable salt thereof, may be able to alleviate the associated ocularinflammation and reverse disease progression or provide symptom relief.

In one aspect, therefore, the disclosure provides a method of treatingan ocular disease in a mammal, the method comprising administering apharmaceutical composition comprising a compound of the disclosure, or apharmaceutically-acceptable salt thereof, or a crystalline form thereof,and a pharmaceutical carrier to the eye of the mammal. In one aspect,the ocular disease is uveitis, diabetic retinopathy, diabetic macularedema, dry eye disease, age-related macular degeneration, retinal veinocclusion or atopic keratoconjunctivitis. In one aspect, the methodcomprises administering the compound of the disclosure, or apharmaceutically acceptable salt thereof, or a crystalline form thereof,by intravitreal injection.

Compounds of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, may also be used in combinationwith one or more compound useful to ocular diseases.

Other Diseases

The compounds of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, may also be useful to treatother diseases such as other inflammatory diseases, autoimmune diseasesor cancers.

The compounds of the disclosure, or a pharmaceutically acceptable saltthereof, or a crystalline form thereof, may be useful to treat one ormore of arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis,transplant rejection, xerophthalmia, psoriatic arthritis, diabetes,insulin dependent diabetes, motor neurone disease, myelodysplasticsyndrome, pain, sarcopenia, cachexia, septic shock, systemic lupuserythematosus, leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, acute lymphoblastic leukemia, acute myelogenousleukemia, ankylosing spondylitis, myelofibrosis, B-cell lymphoma,hepatocellular carcinoma, Hodgkins disease, breast cancer, Multiplemyeloma, melanoma, non-Hodgkin lymphoma, non-small-cell lung cancer,ovarian clear cell carcinoma, ovary tumor, pancreas tumor, polycythemiavera, Sjoegrens syndrome, soft tissue sarcoma, sarcoma, splenomegaly,T-cell lymphoma, and thalassemia major.

Compounds of the disclosure have been demonstrated to be potentinhibitors of the JAK3 enzyme and to be selective for JAK3 over JAK1,JAK2 and TYK2 in enzyme binding assays and to have potent functionalactivity for JAK3 in a cellular assay as described in the followingexamples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

-   -   ACN=acetonitrile    -   Calcd=calculated    -   Boc=tert-Butyloxycarbonyl    -   d=day(s)    -   DIPEA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   EtOAc=ethyl acetate    -   EtOH=ethyl alcohol    -   h=hour(s)    -   HATU=N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   IPA=isopropyl alcohol    -   MeOH=methanol    -   min=minute(s)    -   RT or rt=room temperature    -   SiG=Silica gel    -   TEA=triethylamine    -   THF=tetrahydrofuran    -   THP=tetrahydropyran    -   TFA=trifluoroacetic acid

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD3OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Unless otherwise indicated the following conditions were used forpreparative HPLC purifications.

-   Column: C18, 5 μm 21.2×150 mm or C18, 5 μm 21×250 mm or C14, 5 μm    21×150 mm-   Column temperature: Room Temperature-   Flow rate: 20.0 mL/min-   Mobile Phases: A=Water+0.05% TFA    -   B=ACN+0.05% TFA,-   Injection volume: (100-1500 μL)-   Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 1004 injection with the gradient based on the % B retention of theanalytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Analytic HPLC Conditions Method A Column: LUNA C18 (2), 150×4.60 mm, 3μm

Column temperature: 37° C.Flow rate: 1.0 mL/minInjection volume: 5 μLSample preparation: Dissolve in 1:1 ACN:water

Mobile Phases: A=Water:ACN:TFA (98:2:0.05)

-   -   B=Water:ACN:TFA (2:98:0.05)        Detector wavelength: 250 nm        Gradient: 32 min total (time (min)/% B): 0/2, 10/20, 24/90,        29/90, 30/2, 32/2

Method B Column: LUNA C18 (2), 150×4.60 mm, 3 μm

Column temperature: 37° C.Flow rate: 1.0 mL/minInjection volume: 10 μLSample preparation: Dissolve in 1:1 ACN:water

Mobile Phases: A=Water:ACN:TFA (98:2:0.05)

-   -   B=Water:ACN:TFA (10:90:0.05)        Detector wavelength: 254 nm        Gradient: 35 min total (time (min)/% B): 0/2, 20/25, 23/90,        26/90, 27/2, 35/2

Powder X-ray diffraction patterns were obtained with a Bruker D8-AdvanceX-ray diffractometer using Cu-Kα radiation (λ, =1.54051 Å) with outputvoltage of 45 kV and current of 40 mA. The instrument was operated inBragg-Brentano geometry with incident, divergence, and scattering slitsset to maximize the intensity at the sample. For measurement, a smallamount of powder (5-25 mg) was gently pressed onto a sample holder toform a smooth surface and subjected to X-ray exposure. The samples werescanned in 2θ-2θ mode from 2° to 35° in 2θ with a step size of 0.02° anda scan speed of 0.30° seconds per step. The data acquisition wascontrolled by Bruker DiffracSuite measurement software and analyzed byJade software (version 7.5.1). The instrument was calibrated with acorundum standard, within ±0.02° two-theta angle.

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Analysissoftware. A sample of each crystalline form was accurately weighed intoa covered aluminum pan. After a 5 minute isothermal equilibration periodat 5° C., the sample was heated using a linear heating ramp of 10°C./min from 0° C. to 300° C.

Thermogravimetric analysis (TGA) measurements were performed using a TAInstruments Model Q-50 module equipped with high resolution capability.Data were collected using TA Instruments Thermal Analyst controller andanalyzed using TA Instruments Universal Analysis software. A weighedsample was placed onto a platinum pan and scanned with a heating rate of10° C. from ambient temperature to 300-350° C. The balance and furnacechambers were purged with nitrogen flow during use.

Dynamic moisture sorption (DMS) measurements were performed using a VTIatmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, Fla.33016). A weighed sample was used and the humidity was lowest possiblevalue (close to 0% RH) at the start of the analysis. The DMS analysisconsisted of an initial drying step (0% RH) for 120 minutes, followed bytwo cycles of sorption and desorption with a scan rate of 5% RH/stepover the humidity range of 5% RH to 90% RH. The DMS run was performedisothermally at 25° C.

Preparation 1: 6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole

A mixture of 6-bromo-4-fluoro-1 h-indazole (5 g, 23.25 mmol),3,4-dihydro-2H-pyran (6.38 ml, 69.8 mmol) and p-toluenesulfonic acidmonohydrate (0.442 g, 2.325 mmol) in DCM (76 ml) was stirred at rtovernight. The reaction was concentrated and the resulting residue waspurified by flash column chromatography using a 0-60% EtOAc/Hexanesgradient to yield the desired product (6.08 g, 87% yield).

Preparation 2: tert-butyl3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate

Sodium hydride (1.003 g, 41.8 mmol) was slowly added to a stirringsolution of 1-Boc-Azetidine-3-yl-methanol (6.89 g, 36.8 mmol) in DMF (60mL) under an atmosphere of N₂ at 0° C. and the reaction was allowed towarm to rt. The frothy reaction mixture was stirred for 30 minutes at rtbefore being cooled once again to 0° C. A solution of6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (5.00 g, 16.71mmol) in DMF (20 mL) was slowly cannulated into the reaction mixture,the reaction was warmed to rt and stirred for 2 hours at rt. Thereaction was quenched with the slow addition of H₂O (150 mL) and EtOAc(100 mL) and allowed to stir for 5 minutes. Additional water (100 mL)was added and the biphasic mixture was extracted with EtOAc (3×100 mL).The combined organic fractions were then washed with 1:1 H₂O:brine(3×100 mL) and dried over Na₂SO₄. The resulting clear slightly pink oilwas purified by flash column chromatography using a 0-100% EtOAc/hexanesgradient to yield the desired product as a clear, colorless, viscous oil(7.34 g, 15.74 mmol, 94% yield). (m/z): [M+H]⁺ calcd for C₂₁H₂₈BrN₃O₄466.13 found 466.1.

Preparation 3: tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate

Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethane adduct (1.93 g, 2.36 mmol) was added to a solution of4-hydroxybenzeneboronic acid (3.26 g, 23.61 mmol), tert-butyl3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(7.34 g, 15.74 mmol), and potassium phosphate (10.02 g, 47.2 mmol) in1,4-dioxane (63.0 ml) and water (15.74 ml). The reaction mixture wasdegassed with N₂ for 10 minutes and then stirred at 110° C. for 2 hours.The reaction mixture was concentrated in vacuo to a volume of about 5mL. A saturated aqueous solution of ammonium chloride (20 mL) was addedand the mixture was extracted with methylene chloride (3×20 mL). Themethylene chloride extracts were combined, dried over sodium sulfate andconcentrated in vacuo to yield a brown liquid. The crude liquid waspurified via flash column chromatography using 50% ethyl acetate inhexanes to yield tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(7.55 g, 15.74 mmol, 100% yield). (m/z): [M+H]⁺ calcd for C₂₇H₃₃N₃O₅480.24 found 480.1.

Preparation 4: 4-(4-(azetidin-3-ylmethoxy)-1H-indazol-6-yl)phenol

TFA (10.90 ml) was slowly added to a solution of tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(7.84 g, 16.35 mmol) in dichloromethane (21.80 ml). The clear solutionwas stirred at rt for 5 hours. The reaction was concentrated in vacuo toyield 4-(4-(azetidin-3-ylmethoxy)-1H-indazol-6-yl)phenol as a TFA salt(6.69 g, 16.35 mmol, 100% yield). (m/z): [M+H]⁺ calcd for C₁₇H₁₇N₃O₂296.13 found 296.1.

Example 1A:(E)-4-(dimethylamino)-1-(3-(((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)azetidin-1-yl)but-2-en-1-one

HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, 817 mg, 2.150 mmol) was added to a solution of(E)-4-(dimethylamino)but-2-enoic acid hydrochloride (405 mg, 2.44 mmol)in DMF (2.00 mL) and the reaction mixture was stirred at rt for 5minutes. 4-(4-(azetidin-3-ylmethoxy)-1H-indazol-6-yl)phenol TFA salt(800 mg, 1.954 mmol) was added, followed by DIPEA (1.707 ml, 9.77 mmol)and the reaction mixture was stirred at rt for 15 minutes and thenconcentrated in vacuo to yield a yellow liquid. The crude liquid waspurified via preparatory scale C18 column chromatography using agradient of 20-80% acetonitrile in water with 0.05% trifluoroacetic acidto yield(E)-4-(dimethylamino)-1-(3-(((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)azetidin-1-yl)but-2-en-1-oneas a TFA salt (235.0 mg, 0.578 mmol, 29.6% yield). (m/z): [M+H]⁺ calcdfor C₂₃H₂₆N₄O₃ 407.20 found 407.2. 1H NMR (400 MHz, DMSO-d6) δ 13.07 (s,1H), 10.33 (s, 1H), 7.93 (s, 1H), 7.55 (d, J=7.9 Hz, 2H), 7.21 (S, 1H),6.86 (d, J=8.6 Hz, 2H), 6.79 (s, 1H), 6.66-6.58 (m, 1H), 6.45 (d, J=15.5Hz, 1H), 4.45-4.36 (m, 2H), 4.17-4.07 (m, 2H), 3.91-3.83 (m, 3H),3.22-3.10 (m, 2H), 2.75 (s, 6H).

Example 1B: Crystalline(E)-4-(dimethylamino)-1-(3-(((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)azetidin-1-yl)but-2-en-1-oneForm 4

Step 1

To a suspension of 6-bromo-4-fluoro-1H-indazole (20.0 g, 93 mmol) in 200ml of DCM, was added p-toluenesulfonic acid monohydrate (1.769 g, 9.30mmol). The reaction mixture remained a suspension. 3,4-dihydro-2H-pyran(16.97 ml, 186 mmol) was then added. A complete dissolution of solidswas observed after 5 minutes. The reaction mixture was stirred at RTovernight to form a dark solution. 200 mL of aqueous bicarbonate wereadded, the phases were separated and the organic layer was washed by 200ml of brine and dried with sodium sulfate. The solution was filteredthrough silica plug to remove the dark color and the silica was washedwith 300 ml of DCM. The solvent was evaporated to give 25 g of productas an off-white solid.

Step 2A

In a 500 ml round-bottomed flask was added tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate (22.53 g, 120 mmol) and 100 mlof dimethylacetamide (DMAc). The flask was purged with nitrogen. Cesiumcarbonate (39.2 g, 120 mmol) was added.6-Bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (24 g, 80mmol) was added and the reaction mixture was stirred at 70° C. for 2days. An additional 0.5eq of tert-butyl3-(hydroxymethyl)azetidine-1-carboxylate and cesium carbonate were addedand the reaction was heated at 70° C. overnight to provide fullconversion. The reaction mixture was cooled to RT and then slowly pouredinto stirred ice-cold water (700 ml). The resulting slurry was stirredfor 20 minutes and then filtered and dried to yield 32 g of product. Theproduct was crystallized from methanol-water by adding water slowly asantisolvent until cloudiness. A white slurry developed over time. Thesolid was filtered and dried to yield 28 g of material over 98% pure.

Step 2B

Alternatively, potassium t-butoxide was added to a solution oftert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate in DMAc at 0° C.After 60 minutes,6-Bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole was added at0° C. and the reaction mixture was warmed up to room temperature givingclean complete conversion in less than 6 hours.

Step 3

In a 250 mL Schlenk flast was added tert-butyl3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(10.0 g, 21.44 mmol) and 80 ml of dioxane. (4-Hydroxyphenyl)boronic acid(4.44 g, 32.2 mmol) was added. Potassium phosphate tribasic anhydrous(13.65 g, 64.3 mmol) was added with 20 ml of water and the reactionmixture was purged with nitrogen. PdCl₂(dppf)-CH₂Cl₂ adduct (0.876 g,1.072 mmol) was added and the flask with a condenser was re-filled withnitrogen three times. The reaction mixture was heated at 110° C. for 2 hand 30 minutes to show complete conversion by HPLC. The reaction mixturewas cooled to RT followed by removal of most of the dioxane. 150 ml ofsaturated ammonium chloride was added followed by 150 ml of ethylacetate. The pH was adjusted to neutral with 1M aq HCl. The phases wereseparated and the organic layer was dried with sodium sulfate followedby solvent removal. The crude product was dissolved in 150 ml of DCM andloaded on 300 g SiG column, eluted with 20-50% ethyl acetate in hexanes.The pure fractions were combined and the solvent was evaporated. 100 mlof MeTHF was added, followed by addition of crystalline seeds. A slurrydeveloped over time. 100 ml of DIIPE was added and the slurry wasstirred overnight. Filtration and drying yielded 7.9 g of pure material(>99%).

Step 4

In a 50 mL round-bottomed flask was added tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(85 g, 177 mmol) in 400 ml of methanol. p-Toluenesulfonic acidmonohydrate (101 g, 532 mmol) was added and the reaction mixture wasstirred at RT to give about 90% conversion after 2 days. The reactionmixture was stirred for another 24 hours at room temperature to giveabout 96% conversion. 400 ml of diisopropyl ether was added and theresulting slurry was stirred at room temperature overnight and formed acrystalline salt overnight. Filtration and drying under nitrogen yielded100 g of >99% pure material as a bis PTSA salt.

Step 5

In a 100 mL round-bottomed flask was added(E)-4-(dimethylamino)but-2-enoic acid, HCl (1.899 g, 11.46 mmol) and 20ml of DMF. HCTU o-(1h-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (4.91 g, 11.46 mmol) was added and the reactionmixture was stirred at RT for 20 minutes. In a separate flask was added4-(4-(azetidin-3-ylmethoxy)-1H-indazol-6-yl)phenol, 2TFA (5.0 g, 9.55mmol) and 20 ml of DMF. The resulting solution was cooled to 0° C.,followed by a slow addition of DIPEA (5 ml, 38.2 mmol, 3eq). The abovesolution was added (over 5 minutes) to the pre-activated acid reactionmixture and the reaction mixture was stirred at room temperature for 15minutes. One additional equivalent of DIPEA was added dropwise. Completeconversion was observed by HPLC after 20 minutes at room temperature.The reaction mixture was poured into 200 ml of stirred water. Stickysolids precipitated out at pH around 7. The pH was carefully adjusted toabout 8 with aqueous ammonia. The solution was extracted three timeswith 100 ml of MeTHF. The combined organic layers were dried with sodiumsulfate followed by solvent removal. The crude free base product wasdissolved in 25 ml of 10% methanol in DCM, loaded on 125 g silica gelcolumn and eluted with isocratic 10% methanol in DCM containing 0.5%aqueous ammonia. The pure fractions were combined and the solvent wasevaporated. Recrystallization from acetone yielded >98% pure material(50% yield).

25 mg of compound 1 was suspended in 1 mL of water. The resultingsuspension was stirred for 2 days at 50° C., filtered, washed with 2 mLof water and dried under ambient conditions for a few hours to provideForm 4.

Alternatively, compound 1 was dissolved in ethanol and water or methanoland water by complete dissolution in 10 volumes of alcohol followed byslow addition of approximately 8-10 volumes of water until cloud point.Seeds of Form 4 were added and a slurry developed slowly over time. Thenmore water was added slowly (about 10 volumes) and the solid wasfiltered and dried to give Form 4.

Example 1C: Crystalline(E)-4-(dimethylamino)-1-(3-(((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)azetidin-1-yl)but-2-en-1-oneForm 3

Form 3 is an anhydrous free base crystalline form of compound 1.

150 mg of compound 1 was suspended in 2 mL of a 1:1 mixture ofacetonitrile and isopropanol. The resulting suspension was stirred for 1day at 50° C., filtered, washed with 2 mL of a 1:1 mixture ofacetonitrile and isopropanol and dried under ambient conditions for afew hours to provide Form 3, which was determined to be a crystallineanhydrous free base.

Alternatively, 200 mg of compound 1 as an amorphous free base wasdissolved in 2 ml of IPA at RT. An equal amount of acetonitrile wasadded. More compound (0.5 g total) was added until a saturated solutionformed. Seeds were added and the mixture was stirred overnight. A whiteslurry developed over time. Filtration and drying yielded 400 mg ofproduct as Form 3, which was determined to be an anhydrous free base.

Example 1D: Characterization of Form 3

Samples of Form 3 were analyzed by powder X-ray diffraction (PXRD),differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), and dynamic moisture sorption (DMS).

Powder X-Ray Diffraction

The powder X-ray diffraction pattern of Form 3 is shown in FIG. 9.Observed PXRD two-theta peak positions and d-spacings are shown below.

2-Theta d(Å) Area A % 4.82 18.32 16367 2.7 9.67 9.14 610151 100.0 11.617.61 140214 23.0 11.92 7.42 49009 8.0 12.98 6.81 21804 3.6 13.23 6.6921840 3.6 15.69 5.64 88944 14.6 16.19 5.47 89861 14.7 16.45 5.38 7409112.1 16.67 5.31 57780 9.5 17.61 5.03 158810 26.0 18.88 4.70 261799 42.919.39 4.57 26844 4.4 19.96 4.45 164339 26.9 20.14 4.40 125226 20.5 22.144.01 52100 8.5 23.33 3.81 74095 12.1 23.84 3.73 166452 27.3 24.06 3.70200590 32.9 24.29 3.66 79151 13.0 25.31 3.52 119197 19.5 25.63 3.4788838 14.6 27.06 3.29 62513 10.2 27.31 3.26 47507 7.8 30.10 2.97 184753.0 30.53 2.93 16406 2.7

Thermal Analysis

A representative DSC thermogram of the Form 3 crystalline free form ofthe invention is shown in FIG. 10. The differential scanning calorimetry(DSC) trace recorded at a heating rate of 10° C. per minute exhibits apeak in endothermic heat flow, identified as a melt transition, with anonset at about 197.7° C. and a peak at about 201.3° C. Melting wasfollowed immediately by decomposition.

The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow with a peak at about 201.3° C.

The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow at a temperature between about197.7° C. and about 204° C.

A representative TGA trace of the Form 3 crystalline free form of theinvention is shown in FIG. 11. The thermal gravimetric analysis (TGA)trace of FIG. 11 shows no significant weight loss at temperatures belowthe onset of decomposition at about 195° C.

Dynamic Moisture Sorption Assessment

A representative DMS trace for the Form 3 crystalline free form of theinvention is shown in FIG. 12.

Form 3 demonstrated about 0.33% weight gain in the humidity range of 5%to 90% relative humidity. Form 3 is considered to be non-hygroscopic.

Example 1E: Characterization of Form 4

Samples of Form 4 were analyzed by powder X-ray diffraction (PXRD),differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), and dynamic moisture sorption (DMS).

Powder X-Ray Diffraction

The powder X-ray diffraction pattern of Form 4 is shown in FIG. 13.Observed PXRD two-theta peak positions and d-spacings are shown below.

2-Theta d(Å) Area A % 6.26 14.10 161835 73.90 11.86 7.46 51703 23.6012.51 7.07 82712 37.80 13.16 6.72 72445 33.10 14.98 5.91 53903 24.6016.55 5.35 218849 100.00 16.94 5.23 133498 61.00 17.61 5.03 28496 13.0018.33 4.84 170874 78.10 18.78 4.72 35644 16.30 19.39 4.57 92940 42.5019.57 4.53 86983 39.70 19.84 4.47 19121 8.70 21.45 4.14 22435 10.3021.82 4.07 22934 10.50 22.57 3.94 62931 28.80 23.61 3.76 162063 74.1024.24 3.67 135952 62.10 24.67 3.61 38471 17.60 25.10 3.54 55236 25.2025.39 3.51 32363 14.80 27.19 3.28 27010 12.30 27.39 3.25 32148 14.7028.55 3.12 24879 11.40 31.51 2.84 18975 8.70

Thermal Analysis

A representative DSC thermogram of the Form 4 crystalline free form isshown in FIG. 14. The differential scanning calorimetry (DSC) tracerecorded at a heating rate of 10° C. per minute exhibits a desolvationendotherm with an onset at about 60.9° C. and a peak at about 103.6° C.,and a melting endotherm characterized by an onset at about 167.3° C. Thecompound decomposes at melting and the melting endotherm and thedecomposition exotherm overlap.

A representative TGA trace of the Form 4 crystalline free form is shownin FIG. 15. The thermal gravimetric analysis (TGA) trace of FIG. 15shows a weight loss of about 3.54% at 100° C. The compound desolvates atan onset temperature of about 50° C. The compound decomposes at an onsettemperature of about 165° C.

Dynamic Moisture Sorption Assessment

A representative DMS trace for the Form 4 crystalline free form is shownin FIG. 16.

Form 4 demonstrated about 5.01% weight gain in the humidity range of 5%to 90% relative humidity. Form 4 is considered to be moderatelyhygroscopic.

Preparation 5: tert-butyl(3S)-3-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylate

Sodium hydride (0.201 g, 8.36 mmol) was added to a solution of(S)—N-boc-3-pyridinol (1.25 g, 6.69 mmol) in DMF (12 ml) under anatmosphere of N₂ at 0° C. and the reaction mixture was stirred at rt for20 minutes. 6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(1.0 g, 3.34 mmol) was added and the reaction mixture was stirred at rtfor 1 hour. Water (1 mL) was added and the reaction was concentrated invacuo. The crude material was purified via flash column chromatographyusing 40% ethyl acetate in hexanes to yield tert-butyl(3S)-3-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylate(1.40 g, 3.00 mmol, 90% yield). (m/z): [M+H]⁺ calcd for C₂₁H₂₈BrN₃O₄466.13 found 466.1.

Preparation 6: tert-butyl(3S)-3-((6-(3-chloro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylate

Palladium acetate (0.135 g, 0.600 mmol) was added to a solution of3-chloro-4-hydroxyphenylboronic acid (0.776 g, 4.50 mmol), tert-butyl(3S)-3-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylate(1.40 g, 3.00 mmol), 1,1′-bis(di-t-butylphosphino)ferrocene (0.285 g,0.600 mmol) and potassium phosphate (1.912 g, 9.01 mmol) in 1,4-dioxane(12 ml) and water (3.00 ml). The reaction mixture was degassed withnitrogen for 10 minutes and then stirred at 110° C. for 2 hours. Thereaction mixture was concentrated in vacuo to a volume of about 5 mL. Asaturated aqueous solution of ammonium chloride (20 mL) was added andthe mixture was extracted with methylene chloride (3×20 mL). Themethylene chloride extracts were combined, dried over sodium sulfate andconcentrated in vacuo to yield a brown liquid. The crude liquid waspurified via flash column chromatography using 50% ethyl acetate inhexanes to yield tert-butyl(3S)-3-((6-(3-chloro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylate(1.35 g, 2.63 mmol, 87% yield) as a clear yellow liquid. (m/z): [M+H]⁺calcd for C₂₇H₃₂ClN₃O₅ 514.20 found 514.2.

Preparation 7:(S)-2-chloro-4-(4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenol

4.0N HCl in dioxane (13.13 ml, 52.5 mmol) was added to a solution oftert-butyl(3S)-3-((6-(3-chloro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylate(1.35 g, 2.63 mmol) in dioxane (6 ml), and the reaction mixture wasstirred at 60° C. for 30 minutes. The reaction mixture was concentratedin vacuo to yield(S)-2-chloro-4-(4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenol as an HClsalt (0.962 g, 2.63 mmol, 100% yield). (m/z): [M+H]⁺ calcd forC₁₇H₁₆ClN₃O₂ 330.09 found 330.2.

Example 2:(S)-1-(3-((6-(3-chloro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-en-1-one

N,N-Diisopropylethylamine (4.59 ml, 26.3 mmol) was added to a solutionof (S)-2-chloro-4-(4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenol HClsalt (0.962 g, 2.63 mmol) in DMF (13.0 ml) at 0° C., followed byacryloyl chloride (0.277 ml, 3.41 mmol). The reaction mixture wasstirred at rt for 15 minutes then concentrated to a volume of about 2mL. The crude liquid was purified via preparatory scale C18 columnchromatography using a gradient of 20-80% acetonitrile in water with0.05% trifluoroacetic acid to yield(S)-1-(3-((6-(3-chloro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-en-1-oneas a TFA salt (344 mg, 0.691 mmol, 26.3% yield). (m/z): [M+H]⁺ calcd forC₂₀H₁₈ClN₃O₃ 384.10 found 384.1. 1H NMR (400 MHz, DMSO-d6) δ 13.03 (s,1H), 10.33 (s, 1H), 7.98 (s, 1H), 7.74-7.67 (m, 1H), 7.57-7.49 (m, 1H),7.24 (s, 1H), 7.07 (d, J=0.5 Hz, 1H), 6.80 (s, 1H), 6.69-6.56 (m, 1H),6.18-6.11 (m, 1H), 5.71-5.62 (m, 1H), 4.01-3.60 (m, 5H), 2.35-2.14 (m,2H).

Preparation 8: tert-butyl3-(2-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate

To a dry scintillation vial purged with N₂, tert-butyl3-(2-hydroxypropan-2-yl)azetidine-1-carboxylate (374 mg, 1.74 mmol) wasadded, dissolved in 2.4 mL DMF and the solution was cooled to 0° C. 60%by weight sodium hydride in mineral oil (134 mg, 3.34 mmol) was slowlyadded to the stirring solution and the reaction was allowed to warm tort following the addition. The frothy reaction was stirred for 30minutes before being cooled once again to 0° C. A solution of6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (400 mg, 1.34mmol) in 1 mL DMF was slowly added into the scintillation vialcontaining the organosodium solution. Following the addition, thereaction was warmed to rt and stirred for 2 hours upon which LCMSindicated full conversion of the starting material to the desiredproduct. The reaction was quenched with the slow addition of 1 mL H₂Oand 1 mL EtOAc, which was allowed to stir for 5 minutes. The biphasicsolution was then transferred to a separatory funnel and an additional 5mL H₂O were added. The mixture was extracted with 3 times 10 mL of EtOAcand the aqueous layer was discarded. The combined organic fractions werethen washed with 3×10 mL 1:1 H₂O:brine to remove residual DMF. Theorganic was then dried over Na₂SO₄, filtered, and concentrated to alightly yellow oil. The oil was then purified by flash columnchromatography using a 0-40% EtOAc:hexanes gradient. The product wasisolated pure as a clear, colorless, viscous oil (288 mg, 44% yield).

Preparation 9: tert-butyl3-(2-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate

A 1:1 mixture of Palladium acetate (26.2 mg, 0.116 mmol) and1,1′-Bis(di-t-butylphosphino)ferrocene (55.3 mg, 0.116 mmol) was addedto a solution of 3-chloro-4-hydroxybenzeneboronic acid (151 mg, 0.874mmol), tert-butyl3-(2-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(288 mg, 0.582 mmol), and potassium phosphate (371 mg, 1.747 mmol) in1,4-dioxane (2.0 ml) and water (0.50 ml). The reaction mixture wasdegassed with nitrogen for 10 minutes and then stirred at 110° C. for 2hours. The reaction mixture was concentrated in vacuo to a volume ofabout 5 mL. A saturated aqueous solution of ammonium chloride (5 mL) wasadded and the mixture was extracted with ethyl acetate (2×5 mL). Theethyl acetate extracts were combined, dried over sodium sulfate andconcentrated in vacuo to yield a brown liquid. The crude liquid waspurified via flash column chromatography using 40% ethyl acetate inhexanes to yield tert-butyl3-(2-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(230 mg, 0.424 mmol, 73% yield). (m/z): [M+H]⁺ calcd for C₂₉H₃₆N₃O₅542.24 found 542.3.

Preparation 10:4-(4-((2-azetidin-3-yl)propan-2-yl)oxy)-1-H-indazole-6-yl)-2-chlorophenol

Tert-butyl3-(2-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(230 mg, 0.424 mmol) was dissolved in dichloromethane (1 ml) and TFA (1ml) was slowly added. The clear solution was stirred at rt for 5 hoursupon which LCMS indicated good conversion to the desired product. Thereaction was concentrated down to provide4-(4-((2-azetidin-3-yl)propan-2-yl)oxy)-1-H-indazole-6-yl)-2-chlorophenolas a TFA salt (100% yield). (m/z): [M+H]⁺ calcd for C₁₉H₂₀N₃O₂ 358.13found 358.1.

Example 3A:(E)-1-(3-(2-((6-(3-chloro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidin-1-yl)-4-(dimethylamino)but-2-en-1-one

HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, 100 mg, 0.263 mmol) was added to a solution of(E)-4-(dimethylamino)but-2-enoic acid hydrochloride (34 mg, 0.263 mmol)in DMF (1 mL). The reaction mixture was stirred at rt for 5 minutes then4-(4-((2-azetidin-3-yl)propan-2-yl)oxy)-1-H-indazole-6-yl)-2-chlorophenolTFA salt (118 mg, 0.251 mmol) was added, followed by DIPEA (0.438 ml,2.507 mmol). The reaction mixture was stirred at rt for 15 minutes andthen concentrated in vacuo to yield a yellow liquid. The crude liquidwas purified via preparatory scale SFC (supercritical fluidchromatography) using methanol with liquid carbon dioxide to yield(E)-1-(3-(2-((6-(3-chloro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidin-1-yl)-4-(dimethylamino)but-2-en-1-one(41.3 mg, 0.084 mmol, 33% yield). (m/z): [M+H]⁺ calcd for C₂₅H₂₉ClN₄O₃469.20 found 469.2. ¹H NMR (400 MHz, DMSO-d6) δ 13.07 (s, 1H), 10.33 (s,1H), 7.95 (s, 1H), 7.62 (d, J=2.3 Hz, 1H), 7.47 (dd, J=8.5, 2.3 Hz, 1H),7.34 (S, 1H), 7.04 (d, J=8.5 Hz, 1H), 6.85 (d, J=1.1 Hz, 1H), 6.63-6.53(m, 1H), 6.39 (d, J=15.3 Hz, 1H), 4.37-4.23 (m, 2H), 4.11-3.91 (m, 2H),3.69 (d, J=6.4 Hz, 2H), 3.02-2.90 (m, 1H), 2.62 (s, 9H), 1.32 (S, 9H).

Example 3B: Crystalline(E)-1-(3-(2-((6-(3-chloro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidin-1-yl)-4-(dimethylamino)but-2-en-1-oneForm 1

Form 1 is an anhydrous freebase crystalline form of compound 3.

80 mg of compound 3 as an amorphous freebase was dissolved in 0.5 mL ofethanol. The resulting mixture was stirred for one day at roomtemperature and yielded a precipitate. The solid was isolated byfiltration, washed with 1 mL of ethanol and dried under ambientconditions for a few hours to give Form 1.

Alternatively, in a 50 mL round-bottomed flask, was added compound 3which had been purified by SiG (1.3 g, 2.77 mmol). 10 ml of acetone wasadded and the mixture was stirred at RT. Seeds of crystalline free baseForm 1 were added. A thick white slurry developed over time. Filtrationand drying yielded 1 g of crystalline free base Form 1 with >98% purity.

Example 3B: Crystalline(E)-1-(3-(2-((6-(3-chloro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidin-1-yl)-4-(dimethylamino)but-2-en-1-oneForm 2

Form 2 is a freebase hydrate crystalline form of compound 3.

55 mg of amorphous freebase of compound 3 was dissolved in 0.25 mL ofmethanol. At this stage, water was added as an antisolvent in a ratio ofabout 1:2 methanol:water. The resulting mixture was sonicated forseveral minutes at room temperature to yield a precipitate. Theresulting suspension was stirred for 1 day at room temperature,filtered, washed with 1 mL of methanol and dried to provide Form 2.

Alternatively, compound 3 was dissolved in ethanol and water or methanoland water by complete dissolution in 10 volumes of alcohol followed byslow addition of approximately 8-10 volumes of water until cloud point.Seeds of Form 2 were added and a slurry developed slowly over time. Thenmore water was added slowly (about 10 volumes) and the resulting solidwas filtered and dried to give Form 2.

Example 3C: Characterization of Form 1

Samples of Form 1 were analyzed by powder X-ray diffraction (PXRD),differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), and dynamic moisture sorption (DMS).

Powder X-Ray Diffraction

The powder X-ray diffraction pattern of Form 1 is shown in FIG. 1.Observed PXRD two-theta peak positions and d-spacings are shown below.

2-Theta d(Å) Area A % 5.65 15.63 343682 100.0 7.12 12.40 17888 5.2 10.028.82 13064 3.8 11.16 7.92 60848 17.7 13.10 6.75 13327 3.9 14.22 6.22343837 100.0 14.82 5.97 158470 46.1 15.16 5.84 338146 98.3 16.55 5.3584525 24.6 17.06 5.19 188182 54.7 19.31 4.59 251362 73.1 20.08 4.4257430 16.7 21.08 4.21 29843 8.7 21.65 4.10 45528 13.2 22.51 3.95 8880725.8 22.98 3.87 75161 21.9 24.43 3.64 95427 27.8 25.02 3.56 74549 21.725.72 3.46 24251 7.1 26.80 3.32 23046 6.7 27.06 3.29 26069 7.6 28.313.15 10766 3.1 30.08 2.97 45168 13.1 30.31 2.95 38502 11.2 32.08 2.7922050 6.4

Thermal Analysis

A representative DSC thermogram of the Form 1 crystalline free form ofthe invention is shown in FIG. 2. The differential scanning calorimetry(DSC) trace recorded at a heating rate of 10° C. per minute exhibits apeak in endothermic heat flow, identified as a melt transition, with anonset at about 154.9° C. and a peak at about 162.9° C. Melting wasfollowed immediately by decomposition.

The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow with a peak at about 162.9° C.The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow with a peak at 162.9±3° C.

The crystalline form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow at a temperature between about154.9° C. and about 171° C.

A representative TGA trace of the Form 1 crystalline free form is shownin FIG. 3. The thermal gravimetric analysis (TGA) trace of FIG. 3 showsa small weight loss of about 0.14% at 100° C. The compound decomposes atan onset temperature of about 175° C.

Dynamic Moisture Sorption Assessment

A representative DMS trace for the Form 1 crystalline free form is shownin FIG. 4.

Form 1 demonstrated about 1.62% weight gain in the humidity range of 5%to 90% relative humidity. Form 1 is considered to be slightlyhygroscopic.

Example 3D: Characterization of Form 2

Samples of Form 2 were analyzed by powder X-ray diffraction (PXRD),differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), and dynamic moisture sorption (DMS).

Powder X-Ray Diffraction

The powder X-ray diffraction pattern of Form 2 is shown in FIG. 5.Observed PXRD two-theta peak positions and d-spacings are shown below.

2-Theta d(Å) Area A % 6.90 12.80 248788 52.1 9.15 9.66 73363 15.4 10.008.84 92849 19.4 11.18 7.91 42354 8.9 12.76 6.93 34590 7.2 13.33 6.6433198 6.9 13.82 6.40 75603 15.8 14.43 6.13 20656 4.3 15.51 5.71 9333819.5 16.04 5.52 19890 4.2 17.00 5.21 58922 12.3 17.90 4.95 339924 71.118.31 4.84 477815 100.0 20.90 4.25 169039 35.4 22.06 4.03 68934 14.422.51 3.95 110745 23.2 25.00 3.56 23916 5.0 26.92 3.31 52661 11.0 27.263.27 63506 13.3 27.61 3.23 22650 4.7 29.37 3.04 15298 3.2 30.53 2.9324930 5.2 30.92 2.89 22089 4.6

Thermal Analysis

A representative DSC thermogram of the Form 2 crystalline free form isshown in FIG. 6. The differential scanning calorimetry (DSC) tracerecorded at a heating rate of 10° C. per minute exhibits a desolvationendotherm with an onset at about 52.7° C. and a peak at about 84.4° C.,and a melting endotherm with an onset at about 160.0° C. and a peak atabout 167.6° C. Melting was followed immediately by decomposition.

A representative TGA trace of the Form 2 crystalline free form of theinvention is shown in FIG. 7. The thermal gravimetric analysis (TGA)trace of FIG. 7 shows a weight loss of about 6.73% at 75° C. Thecompound desolvates at an onset temperature of about 25° C. The compounddecomposes at an onset temperature of about 185° C.

Dynamic Moisture Sorption Assessment

A representative DMS trace for the Form 2 crystalline free form of theinvention is shown in FIG. 8. Form 2 converts to a hydrate (Form 2b) atRH above 65%. The dehydration occurs at RH below 15%. The total moistureuptake between 5%-90% RH is 7.99%.

Example 3E: Characterization of Form 2b

Samples of Form 2b were analyzed by powder X-ray diffraction (PXRD).

Powder X-Ray Diffraction

The powder X-ray diffraction pattern of Form 2b is shown in FIG. 17.Observed PXRD two-theta peak positions and d-spacings are shown below.

2-Theta d(Å) Area A % 7.61 11.60 182433 39.1 10.33 8.56 128795 27.611.25 7.86 13807 3 12.71 6.96 102263 21.9 13.23 6.69 34318 7.4 13.666.48 86861 18.6 13.90 6.37 60997 13.1 15.02 5.89 53064 11.4 15.27 5.80166039 35.6 15.88 5.58 163970 35.2 16.33 5.42 87102 18.7 16.76 5.29364312 78.2 17.90 4.95 346920 74.4 18.26 4.85 105152 22.6 20.67 4.29465993 100 21.37 4.16 104882 22.5 21.92 4.05 69905 15 22.31 3.98 13326528.6 22.90 3.88 42178 9.1 23.22 3.83 20810 4.5 23.61 3.76 39278 8.424.74 3.60 160353 34.4 25.78 3.45 67865 14.6 26.23 3.40 30059 6.5 26.733.33 120802 25.9 27.57 3.23 17553 3.8 29.10 3.07 75111 16.1 29.39 3.0497911 21 30.72 2.91 66141 14.2 30.94 2.89 71606 15.4 31.69 2.82 149123.2 32.06 2.79 33582 7.2 33.76 2.65 16247 3.5 34.35 2.61 18479 4

Preparation 11:4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine

4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (2.50 g, 13.23 mmol) was addedto a flask and dissolved in 1,4-dioxane (52.9 ml). p-Toluenesulfonicacid monohydrate (pTsOH, 0.25 g, 1.32 mmol) was then added to the clear,pale yellow solution followed by 3,4-dihydro-2 h-pyran (1.8 ml, 19.84mmol) and the reaction was heated to 40° C. and stirred overnight. Theresulting mixture was then cooled and concentrated in vacuo. The crudewas purified by flash column chromatography using a 0-100% EtOAc:hexanesgradient to yield4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine(2.6 g, 9.52 mmol, 72.0% yield). (m/z): [M+H]⁺ calculated forC₁₀H₁₀Cl₂N₄O 274.0 found 188.9 (loss of THP fragment).

Preparation 12: tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)azetidine-1-carboxylate

To a dry round bottom flask, 1-boc-azetidine-3-yl-methanol (1.96 g,10.47 mmol) was added and dissolved in DMF (20.0 mL). The stirringsolution was cooled to 0° C. and sodium hydride (0.762 g, 19.04 mmol)was then added. The reaction was warmed to room temperature and stirredfor 30 minutes, resulting in a pale pink frothy mixture. The mixture wascooled to 0° C. and a solution of4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine(2.6 g, 9.52 mmol) in DMF (10 mL) was added via cannula. The reactionwas warmed to room temperature and stirred for 3 hours. The reaction wasthen quenched with 120 mL H₂O and extracted 3 times with 30 mL EtOAc.The organic solution was then washed 3 times with 100 mL brine:H₂Osolution (1:1). The organic was dried over Na₂SO₄, filtered, andconcentrated to a pale yellow oil. The crude was purified by flashcolumn chromatography using a 0-50% EtOAc:hexanes gradient to yieldtert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)azetidine-1-carboxylate(1.95 g, 4.60 mmol, 48% yield).

Preparation 13: tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)azetidine-1-carboxylate

In a round bottom flask, tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)azetidine-1-carboxylate(1.95 g, 4.60 mmol) was dissolved in 1,4-dioxane (23.0 ml).4-hydroxyphenylboronic acid (0.95 g, 6.90 mmol) was added followed bywater (7.7 ml). Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(0.56 g, 0.69 mmol) was added and the flask was equipped with a refluxcondenser, set under nitrogen, heated to 110° C., and stirred overnight.The reaction was then cooled to room temperature and the organic solventwas removed in vacuo. H₂O (20 mL) was added to the residue and wasextracted three times with DCM (30 mL). The organic was dried overNa₂SO₄, filtered, and concentrated to a black oil. The crude waspurified by flash column chromatography using a 0-100% EtOAc:hexanesgradient to yield tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)azetidine-1-carboxylate(2.03 g, 4.22 mmol, 92% yield) as a yellow/orange oil. (m/z): [M+H]⁺calculated for C₂₅H₃₁N₅O₅ 482.2 found 482.2.

Preparation 14:4-(4-(azetidin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol

In a round bottom flask, tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)azetidine-1-carboxylate(2.03 g, 4.22 mmol) was dissolved in DCM (10.0 ml) and TFA (5.0 ml) wasslowly added at room temperature. The reaction was stirred for 18 hoursto yield an orange/purple solution. The reaction was concentrated invacuo to yield a grey/orange solid. The title compound was isolated asthe TFA salt in 100% yield (1.25 g, 4.22 mmol). (m/z): [M+H]⁺ calculatedfor C₁₅H₁₅N₅O₂ 298.1 found 298.3.

Example 4:1-(3-(((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)azetidin-1-yl)prop-2-en-1-one

In a round bottom flask,4-(4-(azetidin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenoltrifluoroacetic acid (1.25 g, 4.20 mmol) was dissolved in DMF (21.0 ml)and diisopropylethylamine (3.67 ml, 21.0 mmol) was added. The reactionwas cooled to 0° C. and stirred for 5 minutes before the slow additionof acryloyl chloride (0.273 ml, 3.36 mmol), it was then stirred for 15minutes at 0° C. The reaction was warmed to room temperature andconcentrated in vacuo. The crude was dissolved in 1:1 MeCN:H₂O andpurified by reverse phase preparatory HPLC using a 20-80% MeCN:H₂Ogradient. The desired fractions were combined and lyophilized to givethe title compound (434 mg, 1.235 mmol, 29% yield). (m/z): [M+H]⁺calculated for C₁₈H₁₇N₅O₃ 352.1 found 352.2. ¹H NMR (400 MHz, DMSO-d6) δ8.32-8.23 (d appt, 2H), 8.07 (s, 1H), 6.89-6.81 (d appt, 2H), 6.30 (dd,J=17.0, 10.3 Hz, 1H), 6.07 (dd, J=17.0, 2.3 Hz, 1H), 5.63 (dd, J=10.3,2.3 Hz, 1H), 4.81 (d, J=6.5 Hz, 2H), 4.36 (t, J=8.6 Hz, 1H), 4.12 (dd,J=8.6, 5.4 Hz, 1H), 4.06 (t, 1H), 3.82 (dd, J=10.2, 5.5 Hz, 1H),3.24-3.10 (m, 1H).

Preparation 15: tert-butyl3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate

1-Boc-azetidine-3-yl-methanol (3.44 g, 18.39 mmol) was added to an ovendried flask and dissolved in DMF (41.8 ml). The solution was cooled to0° C. and stirred for 10 minutes before the addition of sodium hydride(0.50 g, 20.89 mmol). The frothy mixture was warmed to room temperatureand stirred for 30 minutes. The reaction was then cooled to 0° C. and asolution of 6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(2.5 g, 8.36 mmol) in DMF (10 mL) was added via cannula. The reactionwas warmed to room temperature and stirred for 1.5 hours. The reactionwas quenched by the addition of 120 mL H₂O and extracted 3 times with 50mL EtOAc. The organic phase was combined and washed 3 times with 100 mLbrine:H₂O solution (1:1). The organic phase was then collected and driedover Na₂SO₄, filtered, and concentrated to a clear, pale yellow oil (3.9g, 8.36 mmol). The crude product was used without any furtherpurification. (m/z): [M+H]⁺ calculated for C₂₁H₂₈BrN₃O₄ 466.14 found466.1.

Preparation 16: tert-butyl3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate

Tert-butyl3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(3.9 g, 8.36 mmol) was added to a flask and dissolved in 1,4-dioxane(44.6 ml). 3-fluoro-4-hydroxyphenylboronic acid (1.96 g, 12.54 mmol) wasadded followed by water (11.2 ml) and potassium phosphate, tribasic(5.33 g, 25.09 mmol).Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (1.02 g, 1.25mmol) was added and the flask was equipped with a reflux condenser andplaced under a nitrogen atmosphere. The reaction was heated to 110° C.and stirred for 18 hours. Once cooled to room temperature, the organicsolvent was removed in vacuo to yield a black oil. To the oil, 10 mL H₂Owas added and the solution was extracted 3 times with 40 mL DCM. Theorganic was collected and dried over Na₂SO₄, filtered, and concentratedto a black oil. The oil was purified via flash column chromatographyusing a 0-100% EtOAc:Hex gradient to yield tert-butyl3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(2.58 g, 5.19 mmol, 62% yield) as a clear yellow oil. (m/z): [M+H]⁺calculated for C₂₇H₃₂FN₃O₅ 498.2 found 498.2.

Preparation 17:4-(4-(azetidin-3-ylmethoxy)-1H-indazol-6-yl)-2-fluorophenol

In a 50 mL round bottom flask, tert-butyl3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(2.58 g, 5.19 mmol) was dissolved in DCM (10.0 ml). TFA (5.00 ml) wasslowly added to the clear yellow solution at room temperature. Thereaction was stirred for 24 hours and the resulting dark yellow/purplesolution was concentrated in vacuo to give the TFA salt of4-(4-(azetidin-3-ylmethoxy)-1H-indazol-6-yl)-2-fluorophenol (3.46 g) asa grey/yellow solid (100% yield). (m/z): [M+H]⁺ calculated forC₁₇H₁₆FN₃O₂ 314.1 found 314.2.

Example 5:(E)-4-(dimethylamino)-1-(3-(((6-(3-fluoro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)azetidin-1-yl)but-2-en-1-one

(2E)-4-(dimethylamino)but-2-enoic acid (464 mg, 2.80 mmol) and HATU (976mg, 2.57 mmol) were added to a round bottom flask, dissolved in DMF (5.5mL) and stirred for 15 minutes. A solution of4-(4-(azetidin-3-ylmethoxy)-1H-indazol-6-yl)-2-fluorophenol (1.0 g, 2.34mmol) in DMF (5.5 mL) was then slowly added to the stirring reactionfollowed by diisopropylethylamine (2.0 ml, 11.70 mmol). The reaction wasstirred for 24 hours and was then concentrated in vacuo. The crude wasdissolved in 1:1 MeCN:H₂O and purified by reverse phase preparatory HPLCusing a 20-80% MeCN:H₂O gradient. The desired fractions were combinedand lyophilized to give the TFA salt of the title compound (271 mg, 0.50mmol, 21% yield). (m/z): [M+H]⁺ calculated for C₂₃H₂₅FN₄O₃ 425.2 found425.2. ¹H NMR (400 MHz, MeOD-d₄) δ 7.94 (d, J=0.8 Hz, 1H), 7.39 (dd,J=12.5, 2.2 Hz, 1H), 7.35-7.28 (m, 1H), 7.24 (t, J=1.0 Hz, 1H),7.03-6.93 (m, 1H), 6.80-6.68 (m, 2H), 6.53 (dt, J=15.2, 1.1 Hz, 1H),4.53 (t, J=8.7 Hz, 1H), 4.47-4.34 (m, 2H), 4.35-4.22 (m, 2H), 4.09 (dd,J=10.8, 5.6 Hz, 1H), 3.93 (dd, J=7.2, 1.1 Hz, 2H), 3.27-3.19 (m, 1H),2.88 (s, 6H).

Preparation 18: 6-Bromo-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole

A mixture of 6-bromo-4-nitro-1H-indazole (3.00 g, 12.4 mmol),3,4-dihydro-2H-pyran, 97% (3.40 ml, 37.2 mmol) and p-toluenesulfonicacid monohydrate (pTsOH, 0.236 g, 1.24 mmol) in DCM (41.3 ml) wasstirred at rt overnight. The reaction was filtered through a pad ofcelite and concentrated. The reaction was assumed to be 100% conversion(4.05 g) and was carried into the subsequent reaction without furtherpurification.

Preparation 19:6-(4-((tert-Butyldimethylsilyl)oxy)phenyl)-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole

A mixture of6-(4-((tert-butyldimethylsilyl)oxy)phenyl)-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(1.00 g, 3.07 mmol), 4-(tert-butyldimethylsilyloxy)phenylboronic acid(1.16 g, 4.60 mmo), and potassium phosphate tribasic (1.30 g, 6.13 mmol)in dioxane (14.9 mL) and H₂O (4.98 mL) was purged with N₂ for 10 mins.[1,1′-bis(diphenylphosphine)ferrocene]dichloropalladium(II) (0.224 g,0.307 mmol) was then added, after which the flask was sealed. Thereaction was heated to 110° C. and stirred for 1 hr. After confirmedfull conversion to the desired product via LCMS, the reaction wasquenched with 20 mL H₂O and 20 mL EtOAc. Both layers were filteredthrough a pad of celite and transferred to a separatory funnel. Themixture was extracted 3 times with 20 mL EtOAc, and the aqueous layerwas discarded. The combined organic fractions were concentrated andsubsequently purified by flash column chromatography using a 0-10%EtOAc:hexanes gradient. The product was isolated pure as a pale-yellowoil (1.03 g, 74.4% yield). (m/z): [M+H]⁺ calcd for C₂₄H₃₁N₃O₄Si 454.61found 454.3.

Preparation 20:6-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-amine

6-(4-((tert-butyldimethylsilyl)oxy)phenyl)-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(0.980 g, 2.16 mmol) and palladium 10% wt on carbon (wet) (0.980 g) weredissolved in THF (2.8 mL) and isopropyl alcohol (11.2 mL). The reactionvessel was back-filled with N₂ for 5 mins, afterwhich it was sealed andplaced under a hydrogen atmosphere. The reaction mixture was left tostir at room temperature for 3 hrs. After LCMS indicated full conversionto the desired product, the reaction mixture with filtered. The filterwas then washed with additional THF. After concentrating and purifyingby flash column chromatography (40 g) using a 0-25% EtOAC:hexanesgradient, the product was isolated (0.562 g, 61.5% yield). (m/z): [M+H]⁺calcd for C₂₄H₃₃N₃O₂Si 424.63 found 424.4.

Preparation 21: tert-butyl(2R)-2-((6-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)carbamoyl)morpholine-4-carboxylate

(R)—N-boc-2-morpholinecarboxylic acid (0.819 g, 3.54 mmol) and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (HATU, 2.69 g, 7.08 mmol) were dissolved inDMF (11.8 mL) and left to stir at room temperature for 5 mins.6-(4-((tert-Butyldimethylsilyl)oxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-amine(1.00 g, 2.36 mmol) was then added, and the reaction mixture was left tostir at room temperature for 2 hrs. LCMS indicated full conversion tothe desired product. The reaction was quenched with 10 mL H₂O and 10 mLDCM. After transfering to a separatory funnel, the mixture was extractedthree times with 10 mL DCM, and the aqueous layer was discarded.Combined organic fractions were concentrated and subsequently purifiedby flash column chromatography (40 g) using a 0-45% EtOAc:hexanesgradient. The products was isolated as a pale-yellow oil (1.01 g, 67%yield). (m/z): [M+H]⁺ calcd for C₃₄H₄₈N₄O₆Si 637.87 found 637.6.

Preparation 22:(R)—N-(6-(4-hydroxyphenyl)-1H-indazol-4-yl)morpholine-2-carboxamide

tert-Buyl(2R)-2-((6-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)carbamoyl)morpholine-4-carboxylate(1.00 g, 1.58 mmol) was dissolved in MeOH (2.0 mL). While stirring, anHCl solution in 4.0M dioxane (12.1 mL, 48.5 mmol) was added slowly. Thereaction mixture was heated to 60° C. and stirred for 30 mins, afterwhich LCMS indicated full conversion to the desired product. The mixturewas concentrated to give the product as an orange oil. The reaction wasassumed to be 100% conversion (0.533 g) and was carried into thesubsequent reaction without further purification.

Example 6:(R)-4-acryloyl-N-(6-(4-hydroxyphenyl)-1H-indazol-4-yl)morpholine-2-carboxamide

(R)—N-(6-(4-hydroxyphenyl)-1H-indazol-4-yl)morpholine-2-carboxamide(0.533 g, 1.58 mmol) was dissolved in DMF (7.9 mL).N,N-diisopropylethylamine (DIPEA, 2.75 mL, 15.8 mmol) was added,followed by slow addition of acryloyl chloride (0.0900 mL, 1.10 mmol).The reaction was stirred at room temperature for 10 mins beforeconversion was monitored by LCMS. After full conversion was indicatedvia LCMS, the reaction mixture was concentrated and purified viareverse-phase HPLC prep using a 10-50% ACN:H₂O gradient. Fractions ofthe desired product were collected and concentrated via lyophilization.The product was isolated (130 mg, 21% yield). (m/z): [M+H]⁺ calcd forC₂₁H₂₀N₄O₄ 393.42 found 393.4. ¹H NMR (400 MHz, Methanol-d4) δ 8.13 (s,1H), 7.74 (d, J=16.3 Hz, 1H), 7.56 (d, J=6.0 Hz, 2H), 7.51 (s, 1H), 6.91(dd, J=8.7, 2.9 Hz, 3H), 6.30 (dt, J=16.8, 2.3 Hz, 1H), 5.84 (d, J=10.6Hz, 1H), 4.45-4.25 (m, 2H), 4.21 (d, J=11.8 Hz, 1H), 4.12-4.02 (m, 1H),3.78 (t, J=11.5 Hz, 1H), 3.53 (m, 1H), 3.29-2.99 (m, 1H).

Preparation 23: tert-butyl(3S)-3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate

Sodium hydride (0.072 g, 3.01 mmol) was added to a solution of(Ss)-N-boc-pyrrolidine-3-methanol (0.444 g, 2.21 mmol) in DMF (6 ml)under an atmosphere of N₂ at 0° C. and the reaction mixture was stirredat rt for 20 minutes.6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (0.600 g, 2.01mmol) was added and the reaction mixture was stirred at rt for 1 hour.Water (1 mL) was added and the reaction was concentrated in vacuo. Thecrude material was purified via flash column chromatography using 40%ethyl acetate in hexanes to yield tert-butyl(3S)-3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate(0.964 g, 1.87 mmol, 93% yield). (m/z): [M+H]⁺ calcd for C₂₂H₃₀BrN₃O₄480.15 found 480.1.

Preparation 24: tert-butyl(3S)-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate

Palladium acetate (0.084 g, 0.375 mmol) was added to a solution of4-hydroxyphenylboronic acid (0.388 g, 2.81 mmol), tert-butyl(3S)-3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate(0.900 g, 1.87 mmol), 1,1′-bis(di-t-butylphosphino)ferrocene (0.178 g,0.375 mmol) and potassium phosphate (1.193 g, 5.62 mmol) in 1,4-dioxane(12 ml) and water (3.00 ml). The reaction mixture was degassed withnitrogen for 10 minutes and then stirred at 110° C. for 2 hours. Thereaction mixture was concentrated in vacuo to a volume of about 5 mL. Asaturated aqueous solution of ammonium chloride (10 mL) was added andthe mixture was extracted with methylene chloride (3×10 mL). Themethylene chloride extracts were combined, dried over sodium sulfate andconcentrated in vacuo to yield a brown liquid. The crude liquid waspurified via flash column chromatography using 50% ethyl acetate inhexanes to yield tert-butyl(3S)-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate(0.800 g, 1.62 mmol, 87% yield) as a clear yellow liquid. (m/z): [M+H]⁺calcd for C₂₈H₃₅N₃O₅ 494.27 found 494.2.

Preparation 25: (S)-4-(4-(pyrrolidin-3-ylmethoxy)-1H-indazol-6-yl)phenol

4.0N HCl in dioxane (8.10 ml, 32.4 mmol) was added to a solution oftert-butyl(3S)-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate(0.800 g, 1.62 mmol) in dioxane (6 ml), and the reaction mixture wasstirred at 60° C. for 30 minutes. The reaction mixture was concentratedin vacuo to yield((S)-4-(4-(pyrrolidin-3-ylmethoxy)-1H-indazol-6-yl)phenol as an HCl salt(0.501 g, 1.62 mmol, 100% yield). (m/z): [M+H]⁺ calcd for C₁₈H₁₉N₃O₂310.16 found 310.3.

Example 7:(S)-1-(3-((6-(3-chloro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-en-1-one

N,N-Diisopropylethylamine (2.83 ml, 16.2 mmol) was added to a solutionof (S)-4-(4-(pyrrolidin-3-ylmethoxy)-1H-indazol-6-yl)phenol HCl salt(0.560 g, 1.62 mmol) in DMF (5 ml) at 0° C., followed by acryloylchloride (0.171 ml, 2.11 mmol). The reaction mixture was stirred at rtfor 15 minutes then concentrated to a volume of about 2 mL. The crudeliquid was purified via preparatory scale C₁₈ column chromatographyusing a gradient of 30-90% acetonitrile in water with 0.05%trifluoroacetic acid to yield(S)-1-(3-(((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)pyrrolidin-1-yl)prop-2-en-1-oneas a TFA salt (226 mg, 0.473 mmol, 29.2% yield). (m/z): [M+H]⁺ calcd forC₂₁H₂₁N₃O₃ 364.17 found 364.2. 1H NMR (400 MHz, DMSO-d6) δ 13.00 (s,1H), 10.31 (s, 1H), 8.04-7.94 (m, 1H), 7.59-7.48 (m, 1H), 7.18 (s, 1H),6.89-6.79 (m, 1H), 6.76-6.70 (m, 1H), 6.65-6.54 (m, 1H), 6.13 (dd,J=16.6, 1.7 Hz, 1H), 5.69-5.62 (m, 1H), 4.25-3.78 (m, 4H), 3.76-3.28 (m,2H), 2.88-2.63 (m, 1H), 2.22-1.72 (m, 2H).

Preparation 26: tert-butyl3-(2-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate

To a dry scintillation vial purged with N₂, tert-butyl3-cyano-3-(hydroxymethyl)azetidine-1-carboxylate (851 mg, 4.01 mmol) wasadded, dissolved in 5 mL DMF and the solution was cooled to 0° C. 60% byweight sodium hydride in mineral oil (267 mg, 6.69 mmol) was slowlyadded to the stirring solution and the reaction was allowed to warm tort following the addition. The frothy reaction was stirred for 30minutes before being cooled once again to 0° C. A solution of6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (800 mg, 2.67mmol) in 2 mL DMF was slowly added into the scintillation vialcontaining the organosodium solution. Following the addition, thereaction was warmed to rt and stirred for 2 hours upon which LCMSindicated full conversion of the starting material to the desiredproduct. The reaction was quenched with the slow addition of 2 mL H₂Oand 2 mL EtOAc, which was allowed to stir for 5 minutes. The biphasicsolution was then transferred to a separatory funnel and an additional10 mL H₂O were added. The mixture was extracted with 3 times 20 mL ofEtOAc and the aqueous layer was discarded. The combined organicfractions were then washed with 3×10 mL 1:1 H₂O:brine to remove residualDMF. The organic was then dried over Na₂SO₄, filtered, and concentratedto a lightly yellow oil. The oil was then purified by flash columnchromatography using a 0-40% EtOAc:hexanes gradient. The product wasisolated pure as a clear, colorless, viscous oil (1.096 g, 83% yield).(m/z): [M+H]⁺ calcd for C₂₂H₂₇BrN₄O₄ 491.13 found 491.1.

Preparation 27: tert-butyl3-cyano-3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate

A 1:1 mixture of Palladium acetate (25.1 mg, 0.112 mmol) and1,1′-Bis(di-t-butylphosphino)ferrocene 98% (53.1 mg, 0.112 mmol) wasadded to a solution of 3-fluoro-4-hydroxybenzeneboronic acid (262 mg,1.679 mmol), tert-butyl3-(2-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(550 mg, 1.119 mmol), and potassium phosphate (713 mg, 3.36 mmol) in1,4-dioxane (4.5 ml) and water (1 ml). The reaction mixture was degassedwith nitrogen for 10 minutes and then stirred at 110° C. for 2 hours.The reaction mixture was concentrated in vacuo. A saturated aqueoussolution of ammonium chloride (5 mL) was added and the mixture wasextracted with ethyl acetate (2×5 mL). The ethyl acetate extracts werecombined, dried over sodium sulfate and concentrated in vacuo to yield abrown liquid. The crude liquid was purified via flash columnchromatography using 40% ethyl acetate in hexanes to yield tert-butyl3-cyano-3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(267 mg, 0.511 mmol, 46% yield). (m/z): [M+H]⁺ calcd for C₂₈H₃₁FN₄O₅523.24 found 523.4.

Preparation 28:3-(((6-(3-fluoro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)azetidine-3-carbonitrile

Tert-butyl3-cyano-3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)methyl)azetidine-1-carboxylate(267 mg, 0.511 mmol) was dissolved in dichloromethane (1 ml) and TFA (1ml) was slowly added. The clear solution was stirred at rt for 5 hoursupon which LCMS indicated good conversion to the desired product. Thereaction was concentrated down to provide3-(((6-(3-fluoro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)azetidine-3-carbonitrileas a TFA salt (100% yield). (m/z): [M+H]⁺ calcd for C₁₈H₁₅FN₄O₂ 339.13found 339.2.

Example 8:(E)-1-(4-(dimethylamino)but-2-enoyl)-3-(((6-(3-fluoro-4-hydroxyphenyl)-1Hindazol-4-yl)oxy)methyl)azetidine-3-carbonitrile

HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium, 121mg, 0.318 mmol) was added to a solution of(E)-4-(dimethylamino)but-2-enoic acid hydrochloride (41.1 mg, 0.318mmol) in DMF (2 mL). The reaction mixture was stirred at rt for 5minutes then3-(((6-(3-fluoro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)methyl)azetidine-3-carbonitrileTFA salt (160 mg, 0.354 mmol) was added, followed by DIPEA (0.618 ml,3.54 mmol). The reaction mixture was stirred at rt for 15 minutes andthen concentrated in vacuo to yield a yellow liquid. The crude liquidwas purified via preparatory scale Zorbax Bonus-RP (2.1×30 mm, 1.8micron) column chromatography using 15-75% acetonitrile in water with0.05% trifluoroacetic acid to yield(E)-1-(4-(dimethylamino)but-2-enoyl)-3-(((6-(3-fluoro-4-hydroxyphenyl)-1Hindazol-4-yl)oxy)methyl)azetidine-3-carbonitrile(56.2 mg, 0.121 mmol, 34% yield). (m/z): [M+H]⁺ calcd for C₂₄H₂₄FN₅O₃450.19 found 450.0. ¹H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 9.77 (s,1H), 7.85 (s, 1H), 7.47 (dd, J=12.8, 2.2 Hz, 1H), 7.31 (dd, J=8.4, 1.7Hz, 1H), 7.22 (S, 1H), 6.99-6.90 (m, 1H), 6.78 (s, 1H), 6.59-6.48 (m,1H), 6.31 (d, J=15.4 Hz, 1H), 4.67-4.60 (m, 3H), 4.40 (d, J=9.1 Hz, 1H),4.27 (d, J=10.5 Hz, 1H), 4.12 (d, J=10.4 Hz, 1H), 3.78 (d, J=6.8 Hz,2H), 2.66 (S, 6H).

Preparation 29: tert-butyl4-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)-4-methylpiperidine-1-carboxylate

To a dry scintillation vial purged with N₂, 1-boc-4-methylpiperidin-4-ol(567 mg, 2.63 mmol) was added, dissolved in 3 mL DMF and the solutionwas cooled to 0° C. 60% by weight sodium hydride in mineral oil (175 mg,4.39 mmol) was slowly added to the stirring solution and the reactionwas allowed to warm to rt following the addition. The frothy reactionwas stirred for 30 minutes before being cooled once again to 0° C. Asolution of 6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(525 mg, 1.755 mmol) in 1 mL DMF was slowly added into the scintillationvial containing the organosodium solution. Following the addition, thereaction was warmed to rt and stirred for 2 hours upon which LCMSindicated full conversion of the starting material to the desiredproduct. The reaction was quenched with the slow addition of 2 mL H₂Oand 2 mL EtOAc, which was allowed to stir for 5 minutes. The biphasicsolution was then transferred to a separatory funnel and an additional10 mL H₂O were added. The mixture was extracted with 3 times 20 mL ofEtOAc and the aqueous layer was discarded. The combined organicfractions were then washed with 3×10 mL 1:1 H₂O:brine to remove residualDMF. The organic was then dried over Na₂SO₄, filtered, and concentratedto a lightly yellow oil. The oil was then purified by flash columnchromatography using a 0-40% EtOAc:hexanes gradient. The product wasisolated pure as a clear, colorless, viscous oil (389 mg, 45% yield).(m/z): [M+H]⁺ calcd for C₂₃H₃₂BrN₃O₄ 494.17 found 494.4.

Preparation 30: tert-butyl4-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)-4-methylpiperidine-1-carboxylate

A 1:1 mixture of Palladium acetate (9.1 mg, 0.040 mmol) and1,1′-Bis(di-t-butylphosphino)ferrocene 98% (19.2 mg, 0.040 mmol) wasadded to a solution of 4-hydroxybenzeneboronic acid (42 mg, 0.30 mmol),tert-butyl4-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)-4-methylpiperidine-1-carboxylate(100 mg, 0.202 mmol), and Potassium phosphate, tribasic, 97%, anhydrous(129 mg, 0.607 mmol) in 1,4-Dioxane (2 ml) and Water (0.5 ml). Thereaction mixture was degassed with nitrogen for 10 minutes and thenstirred at 110° C. for 2 hours. The reaction mixture was concentrated invacuo. A saturated aqueous solution of ammonium chloride (5 mL) wasadded and the mixture was extracted with ethyl acetate (2×5 mL). Theethyl acetate extracts were combined, dried over sodium sulfate andconcentrated in vacuo to yield a brown liquid. The crude liquid waspurified via combiflash column chromatography (12 g) using 40% ethylacetate in hexanes to yield tert-butyl4-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)-4-methylpiperidine-1-carboxylate(96 mg, 0.189 mmol, 94% yield). (m/z): [M+H]⁺ calcd for C₂₉H₃₇N₃O₅5098.28 found 508.3.

Preparation 31:4-(4-((4-methylpiperidin-4-yl)oxy)-1H-indazol-6-yl)phenol

Tert-butyl4-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)-4-methylpiperidine-1-carboxylate(96 mg, 0.189 mmol) was dissolved in 1,4-dioxane (1 ml) and 4N HCl in1,4-dioxane (1.18 ml, 4.73 mmol) was slowly added. The clear solutionwas stirred at rt for 5 hours upon which LCMS indicated good conversionto the desired product. The reaction was concentrated down to provide4-(4-((4-methylpiperidin-4-yl)oxy)-1H-indazol-6-yl)phenol as an HCl salt(100% yield). (m/z): [M+H]⁺ calcd for C₁₉H₂₁N₃O₂ 324.17 found 324.3.

Example 9:(E)-4-(dimethylamino)-1-(4-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)-4-methylpiperidin-1-yl)but-2-en-1-one

HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, 72 mg, 0.189 mmol) was added to a solution of(E)-4-(dimethylamino)but-2-enoic acid hydrochloride (24 mg, 0.186 mmol)in DMF (1 mL). The reaction mixture was stirred at rt for 5 minutes then4-(4-((4-methylpiperidin-4-yl)oxy)-1H-indazol-6-yl)phenol HCl salt (68.0mg, 0.189 mmol) was added, followed by DIPEA (0.330 ml, 1.890 mmol). Thereaction mixture was stirred at rt for 15 minutes and then concentratedin vacuo to yield a yellow liquid. The crude liquid was purified viapreparatory scale Zorbax Bonus-RP (2.1×30 mm, 1.8 micron) columnchromatography using 5-65% acetonitrile in water with 0.05%trifluoroacetic acid to yield(E)-4-(dimethylamino)-1-(4-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)-4-methylpiperidin-1-yl)but-2-en-1-one(39.0 mg, 0.063 mmol, 34% yield). (m/z): [M+H]⁺ calcd for C₂₅H₃₀FN₄O₃435.24 found 435.2. ¹H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 9.66 (s,1H), 8.02 (d, J=0.9 Hz, 1H), 7.52-7.44 (m, 2H), 7.27 (s, 1H), 6.90 (d,J=15.0 Hz, 1H), 6.86-6.78 (m, 3H), 6.58-6.47 (m, 1H), 3.99-3.86 (m, 1H),3.83 (d, J=7.2 Hz, 2H), 3.78-3.55 (m, 2H), 2.74 (s, 6H), 1.40 (s, 3H).

Preparation 32:4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine

3,4-Dihydro-2 h-pyran (2.0 g, 24.07 mmol) was added to a solution of4,6-dichloro-1H-pyrazolo[3,4-b]pyridine (2.50 g, 13.23 mmol) inmethylene chloride (30 mL), followed by p-toluenesulfonic acidmonohydrate (0.275 g, 1.60 mmol), and the reaction mixture was stirredat rt for 1 h. Water (50 mL) was added and the mixture was extractedwith methylene chloride (3×50 mL). The methylene chloride extracts werecombined, washed with brine (1×50 mL), dried over sodium sulfate andconcentrated to yield a clear yellow liquid. The crude liquid waspurified via flash column chromatography using 10% EtOAc in hexanes toyield4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine(3.0 g, 11.1 mmol, 67% yield). (m/z): [M+H]⁺ calculated for C₁₁H₁₁C₁₂N₃O272.04 found 272.21.

Preparation 33: tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate

NaH (60% dispersion in mineral oil, 160 mg, 4.0 mmol) was added to asolution of 1-boc-azetidine-3-yl-methanol (760 mg, 4.0 mmol) in DMF (5mL) and diethyl ether (5 mL) at 0° C. and the reaction mixture wasstirred at 0° C. for 15 minutes.4,6-Dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine(1.0 g, 3.7 mmol) was added and the reaction mixture was stirred at rtfor 1 h. Water (10 mL) was added and the reaction mixture was extractedwith methylene chloride (3×10 mL). The methylene chloride extracts werecombined, washed with brine (1×10 mL), dried over sodium sulfate andconcentrated in vacuo to yield a clear yellow liquid. The crude liquidwas purified via flash column chromatography using 30% EtOAc in hexanesto yield tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate(1.1 g, 2.60 mmol, 70% yield). (m/z): [M+H]⁺ calculated for C₂₀H₂₇ClN₄O₄423.18 found 423.25.

Preparation 34: tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate

A solution of sodium carbonate (500 mg, 4.72 mmol) in water (4 mL) wasadded to 4-hydroxyphenylboronic acid (392 mg, 2.84 mmol) and tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate(1.0 g, 2.36 mmol) in dioxane (20 mL) and the reaction mixture wasdegassed with nitrogen for 10 minutes. PdCl₂(dppf).DCM (196 mg, 0.24mmol) was added and the reaction mixture was degassed further for 5minutes and then stirred at 110° C. for 4 hours. The reaction mixturewas cooled to rt and then filtered through a bed of Celite. The filteredmaterial was washed with methylene chloride and the filtrates werecombined and washed with water (1×10 mL), brine (1×10 mL), dried oversodium sulfate and concentrated in vacuo to yield a clear brown liquid.The crude liquid was purified via flash column chromatography using 50%EtOAc in hexanes to yield tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate(1.02 g, 2.12 mmol, 76% yield). (m/z): [M+H]⁺ calculated for C₂₆H₃₂N₄O₅481.25 found 481.65.

Preparation 35:4-(4-(azetidin-3-ylmethoxy)-1H-pyrazolo[3,4-b]pyridin-6-yl)phenol

TFA (4.0 ml) was added slowly to a solution of tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate(500 mg, 1.04 mmol) in methylene chloride (10 mL) at 0° C. and thereaction mixture was stirred at rt for 3 hours. The reaction mixture wasconcentrated in vacuo and the resulting solid was triturated with 5%methanol:methylene chloride to yield the title compound as the TFA salt(330 mg, 0.804 mmol, 77% yield). (m/z): [M+H]⁺ calculated for C₁₆H₁₆N₄O₂297.14 found 297.15.

Example 10:1-(3-(((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidin-1-yl)prop-2-en-1-one

To a solution of4-(4-(azetidin-3-ylmethoxy)-1H-pyrazolo[3,4-b]pyridin-6-yl)phenol TFAsalt (35.5 mg, 0.087 mmol) in DMF (1 mL) was added DIPEA (0.151 ml,0.865 mmol), followed by the addition of acryloyl chloride (7.0 uL,0.087 mmol). The reaction mixture was stirred at rt for 15 minutes andthen concentrated in vacuo to yield a yellow liquid. The crude liquidwas purified via preparatory scale Zorbax Bonus-RP (2.1×30 mm, 1.8micron) column chromatography using 5-75% acetonitrile in water with0.05% trifluoroacetic acid to yield1-(3-(((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidin-1-yl)prop-2-en-1-one(13.4 mg, 0.028 mmol, 33% yield). (m/z): [M+H]⁺ calcd for C₁₉H₂₈N₄O₃351.15 found 351.1. ¹H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 9.80 (s,1H), 7.98 (d, J=8.9 Hz, 3H), 7.13 (s, 1H), 6.85 (d, J=8.7 Hz, 2H), 6.32(dd, J=17.0, 10.3 Hz, 1H), 6.09 (dd, J=17.0, 2.3 Hz, 1H), 5.64 (dd,J=10.3, 2.3 Hz, 1H), 4.55 (d, J=6.7 Hz, 1H), 4.38 (t, J=8.5 Hz, 1H),4.15-4.02 (m, 2H), 3.80 (dd, J=10.0. 5.4 Hz, 2H), 3.22-3.07 (m, 1H).

Preparation 36:6-bromo-4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole

3,4-Dihydro-2 h-pyran (19.79 mL, 216.5 mmol) was added to a solution of6-bromo-4-chloro-1H-indazole (10.0 g, 43.3 mmol) in ethyl acetate (200mL) at 0° C., followed by p-toluenesulfonic acid monohydrate (1.64 g,8.66 mmol), and the reaction mixture was stirred at 50° C. for 4 h.Saturated aqueous sodium bicarbonate (100 mL) was added and the mixturewas extracted with ethyl acetate (3×100 mL). Ethyl acetate extracts werecombined, washed with water (1×100 mL), brine (1×100 mL), dried oversodium sulfate and concentrated to yield a yellow solid. The crudeliquid was purified via trituration in n-pentane to yield6-bromo-4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (13.0 g, 41.2mmol, 95% yield). (m/z): [M+H]⁺ calculated for C₁₂H₁₂BrClN₂O 314.99found 314.93.

Preparation 37:4-(4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)phenol

A solution of sodium carbonate (8.77 g, 82.8 mmol),4-hydroxyphenylboronic acid (6.85 g, 49.7 mmol) and6-bromo-4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (13.0 g, 41.4mmol) in dioxane (200 mL) and water (50 mL) was degassed with nitrogenfor 15 minutes. PdCl₂(dppf).DCM (3.37 g, 4.14 mmol) was added and thereaction mixture was degassed further for 5 minutes and then stirred at125° C. for 2 hours. The reaction mixture was cooled to rt, ethylacetate was added and the reaction mixture was filtered through a bed ofCelite. Water was added (100 mL) and the mixture was extracted withethyl acetate. The ethyl acetate extracts were combined, dried oversodium sulfate and concentrated in vacuo to yield a clear brown liquid.The crude liquid was purified via flash column chromatography using a0-30% EtOAc in hexanes gradient to yield4-(4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)phenol (7.5 g,22.9 mmol, 55% yield). (m/z): [M+H]⁺ calculated for C₁₈H₁₇ClN₂O₂ 329.11found 329.08.

Preparation 38: tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)(methyl)amino)methyl)azetidine-1-carboxylate

A 1:2 mixture of palladium acetate (68.3 mg, 0.304 mmol) and2-(di-t-butylphosphino)biphenyl (182.0 mg, 0.608 mmol) was added to asolution of4-(4-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)phenol (500 mg,1.521 mmol), tert-butyl 3-((methylamino)methyl)azetidine-1-carboxylate(457 mg, 2.281 mmol), and sodium tert-butoxide, >99.9% (731 mg, 7.60mmol) in toluene (15 ml). The reaction mixture was degassed withnitrogen for 10 minutes and then stirred at 110° C. for 16 hours. Thereaction mixture was concentrated in vacuo to a volume of about 5 mL. Asaturated aqueous solution of ammonium chloride (5 mL) was added and themixture was extracted with ethyl acetate (2×5 mL). The ethyl acetateextracts were combined, dried over sodium sulfate and concentrated invacuo to yield a brown liquid. The crude liquid was purified viacombiflash column chromatography (24 g) using 50% ethyl acetate inhexanes to yield tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)(methyl)amino)methyl)azetidine-1-carboxylate(368 mg, 0.747 mmol, 49% yield). (m/z): [M+H]⁺ calcd for C₂₈H₃₆N₄O₄493.28 found 493.4.

Preparation 39:4-(4-((azetidin-3-yl)methyl)(methyl)amino)-1H-indazol-6-yl)phenol

Tert-butyl3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)(methyl)amino)methyl)azetidine-1-carboxylate(368 mg, 0.747 mmol) was dissolved in Dichloromethane (1.5 ml) and TFA(1.5 ml) was slowly added. The clear solution was stirred at rt for 5hours upon which LCMS indicated good conversion to the desired product.The reaction was concentrated down to provide4-(4-((azetidin-3-ylmethyl)(methyl)amino)-1H-indazol-6-yl)phenol as aTFA salt (100% yield). (m/z): [M+H]⁺ calcd for C₁₈H₂₀N₄O 309.17 found309.2.

Example 11:(E)-4-(dimethylamino)-1-(3-(((6-(4-hydroxyphenyl)-1H-indazol-4-yl)(methyl)amino)methyl)azetidin-1-yl)but-2-en-1-one

HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, 59.5 mg, 0.157 mmol) was added to a solution of(E)-4-(dimethylamino)but-2-enoic acid hydrochloride (34 mg, 0.263 mmol)in DMF (1 mL). The reaction mixture was stirred at rt for 5 minutes then4-(4-((azetidin-3-ylmethyl)(methyl)amino)-1H-indazol-6-yl)phenol TFAsalt (63 mg, 0.149 mmol) was added, followed by DIPEA (0.260 ml, 1.491mmol). The reaction mixture was stirred at rt for 15 minutes and thenconcentrated in vacuo to yield a yellow liquid. The crude liquid waspurified via preparatory scale Zorbax Bonus-RP (2.1×30 mm, 1.8 micron)column chromatography using 5-65% acetonitrile in water with 0.05%trifluoroacetic acid to yield(E)-4-(dimethylamino)-1-(3-(((6-(4-hydroxyphenyl)-1H-indazol-4-yl)(methyl)amino)methyl)azetidin-1-yl)but-2-en-1-one(47.6 mg, 0.086 mmol, 96% yield). (m/z): [M+H]⁺ calcd for C₂₄H₂₉N₅O₂420.24 found 420.2.

Preparation 40: tert-butyl(3S)-3-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)thio)pyrrolidine-1-carboxylate

To a stirred solution of6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (600 mg, 2.0mmol) and tert-butyl (S)-3-mercaptopyrrolidine-1-carboxylate (488 mg,2.20 mmol) in DMSO (5 mL) was added Cs₂CO₃ (1.30 mg, 4.0 mmol). Thereaction mixture was stirred at 120° C. for 16 hours. Water was addedand the reaction mixture was extracted with EtOAc (3×50 ml). The EtOAcextracts were combined, washed with water followed by brine, dried overanhydrous sodium sulphate, filtered and concentrated in vacuo to get thecrude intermediate product as clear viscous liquid.

To a stirred solution of the crude intermediate in dioxane (20 mL) andwater (4 ml) was added 4-hydroxyphenylboronic acid (240 mg, 1.74 mmol)and Na₂CO₃ (461 mg, 4.35 mmol) and the reaction mixture was degassedwith nitrogen for 15 minutes. Pd(dppf)Cl₂.DCM (118 mg, 0.0145 mmol) wasadded and the reaction mixture was degassed further for 5 minutes andthen stirred at 110° C. for 4 hours. The reaction mixture was cooled tort, water was added and the reaction mixture was extracted with ethylacetate (3×50 mL). The ethyl acetate extracts were combined, dried oversodium sulfate and concentrated in vacuo to yield a clear brown liquid.The crude liquid was purified via flash column chromatography using a30-50% EtOAc in hexanes gradient to yieldtert-butyl(3S)-3-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)thio)pyrrolidine-1-carboxylate(600 mg, 1.21 mmol, 61%). (m/z): [M+H]⁺ calculated for C₂₇H₃₃ClN₃O₄S496.23 found 496.42.

Preparation 41: (S)-4-(4-(pyrrolidin-3-ylthio)-1H-indazol-6-yl)phenol

4.0N HCl in dioxane (10.0 ml, 40.0 mmol) was added to a solution oftert-butyl(3S)-3-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)thio)pyrrolidine-1-carboxylate(400 mg, 0.808 mmol) in methanol (6 ml), and the reaction mixture wasstirred at 40° C. for 16 h. The reaction mixture was concentrated invacuo and the resulting residue was purified via preparatory scale C18column chromatography using a gradient of 20-80% acetonitrile in waterwith 0.05% trifluoroacetic acid to yield to yield(S)-4-(4-(pyrrolidin-3-ylthio)-1H-indazol-6-yl)phenol as an TFA salt(175 mg, 0.411 mmol, 51% yield). (m/z): [M+H]⁺ calcd for C₁₇H₁₇N₃OS312.12 found 312.22.

Example 12:(S)-1-(3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)thio)pyrrolidin-1-yl)prop-2-en-1-one

N,N-Diisopropylethylamine (0.079 ml, 0.450 mmol) was added to a solutionof (S)-4-(4-(pyrrolidin-3-ylthio)-1H-indazol-6-yl)phenol TFA salt (38.5mg, 0.091 mmol) in DMF (0.2 ml) at 0° C., followed by acryloyl chloride(8.0 μL, 0.099 mmol). The reaction mixture was stirred at rt for 15minutes and the crude liquid was purified via preparatory scale C₁₈column chromatography using a gradient of acetonitrile in water with0.05% trifluoroacetic acid to yield(S)-1-(3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)thio)pyrrolidin-1-yl)prop-2-en-1-oneas a TFA salt (17.1 mg, 0.046 mmol, 51% yield). (m/z): [M+H]⁺ calcd forC₂₀H₁₉N₃O₂S 366.13 found 366.1.

Preparation 42: tert-butyl(3S)-3-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)sulfonyl)pyrrolidine-1-carboxylate

Basic alumina (200 mg) and Oxone (744 mg, 2.42 mmol) were added to asolution of tert-butyl(3S)-3-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)thio)pyrrolidine-1-carboxylate(400 mg, 0.80 mmol) in chloroform (50 mL) and water (1 mL) and thereaction mixture was stirred at 65° C. for 12 h. The reaction mixturewas filtered through a bed of Celite, and the filtered material waswashed with chloroform. The filtrates were combined, water was added andthe mixture was extracted with chloroform (3×100 mL). The chloroformextracts were combined, washed with brine, dried over sodium sulfate andconcentrated in vacuo. The crude liquid was purified via flash columnchromatography using a 30% EtOAc in hexanes to yield tert-butyl(3S)-3-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)sulfonyl)pyrrolidine-1-carboxylate(200 mg, 0.379 mmol, 47% yield). (m/z): [M+H]⁺ calculated forC₂₇H₃₃ClN₃O₆ 528.22 found 528.48.

Preparation 43:(S)-4-(4-(pyrrolidin-3-ylsulfonyl)-1H-indazol-6-yl)phenol

4.0N HCl in dioxane (10.0 ml, 40.0 mmol) was added to a solution of(3S)-3-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)sulfonyl)pyrrolidine-1-carboxylate(200 mg, 0.379 mmol) in methanol (2 ml), and the reaction mixture wasstirred at rt for 8 h. The reaction mixture was concentrated in vacuoand the resulting residue was triturated with diethyl ether to yield((S)-4-(4-(pyrrolidin-3-ylsulfonyl)-1H-indazol-6-yl)phenol as an HClsalt (140 mg, 0.369 mmol, 97% yield). (m/z): [M+H]⁺ calcd forC₁₇H₁₇N₃O₃S 344.11 found 344.04.

Example 13:((S)-1-(3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)sulfonyl)pyrrolidin-1-yl)prop-2-en-1-one

N,N-Diisopropylethylamine (0.079 ml, 0.450 mmol) was added to a solutionof (((S)-4-(4-(pyrrolidin-3-ylsulfonyl)-1H-indazol-6-yl)phenol HCl salt(31.0 mg, 0.082 mmol) in DMF (0.2 ml) at 0° C., followed by acryloylchloride (8.0 μL, 0.099 mmol). The reaction mixture was stirred at rtfor 10 minutes and the crude liquid was purified via preparatory scaleC₁₈ column chromatography using a gradient of acetonitrile in water with0.05% trifluoroacetic acid to yield((S)-1-(3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)sulfonyl)pyrrolidin-1-yl)prop-2-en-1-oneas a TFA salt (3.1 mg, 0.0061 mmol, 7% yield). (m/z): [M+H]⁺ calcd forC₂₀H₁₉N₃O₄S 398.12 found 398.0.

Preparation 44: tert-butyl4-((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)methyl)piperidine-1-carboxylate

A 0.5 M solution of 9-BBN in THF (7.92 mL, 3.96 mmol) was added totert-butyl 4-methylenepiperidine-1-carboxylate (468 mg, 2.37 mmol) andthe reaction mixture was stirred at 80° C. for 2 h. The reaction mixturewas cooled to rt and then cannulated into a preformed solution of4-bromo-6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (500 mg, 1.58mmol), potassium carbonate (548 mg, 3.96 mmol) and Pd(dppf)C12.DCM (129mg, 0.15 mmol) in DMF (10.0 ml) & H₂O (2.0 ml). The reaction mixture wasstirred at 80° C. for 16 h. The reaction mixture was filtered through abed of Celite, and the filtered material was washed with ethyl acetate.The filtrates were combined, water (100 mL) was added and the mixturewas extracted with ethyl acetate (2×150 mL). The cethyl acetate extractswere combined, washed with brine, dried over sodium sulfate andconcentrated in vacuo. The crude liquid was purified via flash columnchromatography using a 30% EtOAc in hexanes to yield tert-butyl4-((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)methyl)piperidine-1-carboxylate(460 mg, 1.06 mmol, 67% yield). (m/z): [M+H]⁺ calculated forC₂₃H₃₂ClN₃O₃ 434.22 found 434.47.

Preparation 45: tert-butyl4-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)methyl)piperidine-1-carboxylate

4-Hydroxyphenylboronic acid (195 mg, 1.41 mmol) was added to asuspension of tert-butyl4-((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)methyl)piperidine-1-carboxylate(410 mg, 0.94 mmol) in dioxane (10.0 mL) and water (2.0 mL), followed bypotassium phosphate (401 mg, 1.88 mmol) and the reaction mixture wasdegassed with nitrogen for 15 minutes.2′-(Dimethylamino)-2-biphenylyl-palladium(II) chloridedinorbornylphosphine complex (52 mg, 0.07 mmol) was added and thereaction mixture was heated under microwave irradiation at 100° C. for 1hour. The reaction mixture was cooled to rt and then filtered through abed of Celite. Water (100 mL) was added to the filtrate, which was thenextracted with ethyl acetate (3×100 mL). The ethyl acetate extracts werecombined, washed with water, brine, dried over sodium sulfate andconcentrated in vacuo. The crude liquid was purified via flash columnchromatography using 50% EtOAc in hexanes to yield tert-butyl4-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)methyl)piperidine-1-carboxylate(300 mg, 0.610 mmol, 65%). (m/z): [M+H]⁺ calculated for C₂₉H₃₇N₃O₄492.29 found 492.46.

Preparation 46: 4-(4-(piperidin-4-ylmethyl)-1H-indazol-6-yl)phenol

4.0N HCl in dioxane (10.0 ml, 40.0 mmol) was added to a solution oftert-butyl4-((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)methyl)piperidine-1-carboxylate(300 mg, 0.610 mmol) in methanol (2 ml), and the reaction mixture wasstirred at rt for 8 h. The reaction mixture was concentrated in vacuoand the resulting residue was triturated with diethyl ether to yield4-(4-(piperidin-4-ylmethyl)-1H-indazol-6-yl)phenol as an HCl salt (195mg, 0.567 mmol, 93% yield). (m/z): [M+H]⁺ calcd for C₁₉H₂₁N₃O 308.18found 308.10.

Example 14:1-(4-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)methyl)piperidin-1-yl)prop-2-en-1-one

N,N-Diisopropylethylamine (0.079 ml, 0.450 mmol) was added to a solutionof 4-(4-(piperidin-4-ylmethyl)-1H-indazol-6-yl)phenol HCl salt (27.5 mg,0.080 mmol) in DMF (0.2 ml) at 0° C., followed by acryloyl chloride(8.00 μL, 0.099 mmol). The reaction mixture was stirred at rt for 10minutes and the crude liquid was purified via preparatory scale C18column chromatography using a gradient of acetonitrile in water with0.05% trifluoroacetic acid to yield1-(4-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)methyl)piperidin-1-yl)prop-2-en-1-oneas a TFA salt (7.2 mg, 0.015 mmol, 19% yield). (m/z): [M+H]⁺ calcd forC₂₂H₂₃N₃O₂ 362.19 found 362.1.

Preparation 47: tert-butyl(R,E)-2-(3-ethoxy-3-oxoprop-1-en-1-yl)pyrrolidine-1-carboxylate

Ethyl 3-(triphenylphosphoranylidene)propanoate (3.64 g, 10.0 mmol) wasadded to a solution of tert-butyl (R)-2-formylpyrrolidine-1-carboxylate(2.0 g, 10.0 mmol) in methylene chloride (40 mL) at 0° C. and thereaction mixture was stirred at rt for 4 h. Water (200 mL) was added andthe reaction mixture was extracted with methylene chloride (2×100 mL).The methylene chloride extracts were combined, dried over sodium sulfateand concentrated in vacuo. The crude liquid was purified via flashcolumn chromatography using 5% ethyl acetate in hexanes to yieldtert-butyl(R,E)-2-(3-ethoxy-3-oxoprop-1-en-1-yl)pyrrolidine-1-carboxylate (1.70 g,6.31 mmol, 63% yield).

Preparation 48: ethyl (R,E)-3-(1-methylpyrrolidin-2-yl)acrylate

4M HCl in dioxane (7.20 mL, 28.8 mmol) was added to a solution of TFA(10.90 ml) was slowly added to a solution of tert-butyl(R,E)-2-(3-ethoxy-3-oxoprop-1-en-1-yl)pyrrolidine-1-carboxylate (1.70 g,6.31 mmol) in dioxane (40 mL) and the reaction mixture was stirred at rtfor 2 h. The reaction was concentrated in vacuo to yield the crudeintermediate. A 37% solution of formaldehyde in water (4.19 mL, 41.4mmol) was added to a solution of the crude intermediate in methylenechloride (50 mL) at 0° C., followed by sodium triacetoxyborohydride(5.26 g, 24.8 mmol), and the reaction mixture was stirred at rt for 16h. Ice cold water (20 mL) was added and the reaction mixture wasextracted with methylene chloride (2×100 mL). The methylene chlorideextracts were combined, washed with brine, dried over sodium sulfate andconcentrated in vacuo to yield ethyl(R,E)-3-(1-methylpyrrolidin-2-yl)acrylate (0.900 g, 4.915 mmol, 78%yield). (m/z): [M+H]⁺ calcd for C₁₀H₁₇NO₂ 184.14 found 184.0.

Preparation 49: (R,E)-3-(1-methylpyrrolidin-2-yl)acrylic acid

Sodium hydroxide (294 mg, 7.36 mmol) was added to a solution of ethyl(R,E)-3-(1-methylpyrrolidin-2-yl)acrylate (0.900 g, 4.915 mmol, 78%yield) in 2:1 THF/water (15 mL) and the reaction mixture was stirred atrt for 4 h. The reaction mixture was concentrated in vacuo to yieldethyl (R,E)-3-(1-methylpyrrolidin-2-yl)acrylic acid (668 mg, 4.30 mmol,88% yield). (m/z): [M+H]⁺ calcd for C₈H₁₃NO₂ 156.10 found 156.26.

Example 15:(E)-1-((S)-3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)-3-((R)-1-methylpyrrolidin-2-yl)prop-2-en-1-one

A solution of HATU (0.031 g, 0.083 mmol) in DMF (0.1 mL) was added toethyl (R,E)-3-(1-methylpyrrolidin-2-yl)acrylic acid (14.0 mg, 0.090mmol), followed by a solution of(S)-4-(4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenol hydrochloride(0.025 g, 0.075 mmol) and DIPEA (0.065 ml, 0.375 mmol), the reactionmixture was stirred at rt for 15 minutes and the crude liquid waspurified via preparatory scale C18 column chromatography using agradient of acetonitrile in water with 0.05% trifluoroacetic acid toyield((E)-1-((S)-3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)-3-((R)-1-methylpyrrolidin-2-yl)prop-2-en-1-oneas a TFA salt (7.8 mg, 0.014 mmol, 19% yield). (m/z): [M+H]⁺ calcd forC₂₅H₂₈N₄O₃ 433.23 found 433.1.

Preparation 50: tert-butyl 2-((dimethylamino)methyl)acrylate

Paraformaldehyde (1.20 g, 43.2 mmol) was added to a solution of Malonicacid (2.0 g, 19.3 mmol) in 1,4-dioxane (20 mL), followed by a 2Msolution of dimethylamine in THF (9.60 mL, 19.2 mmol) and the reactionmixture was stirred at 70° C. for 1 h. The reaction mixture wasconcentrated in vacuo and the crude product was recrystallized usingdiethyl ether and acetone to yield the intermediate as a white solid.The intermediate white solid was dissolved in t-BuOH (100 mL) anddi-tert-butyl dicarbonate (4.60 mL, 20.7 mmol) was added to the reactionmixture followed by 4-dimethylaminopyridine (511 mg, 4.18 mmol) and thereaction mixture was stirred at rt for 4 h. Methylene chloride (500 mL)was added and the reaction mixture was washed with water (2×500 mL),dried over sodium sulfate and concentrated in vacuo. The crude residuewas purified via flash column chromatography using 2% methanol inmethylene chloride to yield tert-butyl 2-((dimethylamino)methyl)acrylate(220 mg, 1.19 mmol, 6% yield).

Preparation 51: 2-((dimethylamino)methyl)acrylic acid

A 1N aqueous solution of HCl (5.0 mL, 5 mmol) was added to tert-butyl2-((dimethylamino)methyl)acrylate (220 mg, 1.19 mmol) and the reactionmixture was stirred at 100° C. for 15 minutes. The reaction mixture wasconcentrated in vacuo and the resulting residue was azeotroped withtoluene and further triturated with diethyl ether to yield2-((dimethylamino)methyl)acrylic acid as a HCl salt (119 mg, 0.719 mmol,60% yield). (m/z): [M+H]⁺ calcd for C₆H₁₁NO₂ 130.09 found 130.22.

Example 16:(S)-2-((dimethylamino)methyl)-1-(3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-en-1-one

A solution of HATU (0.031 g, 0.083 mmol) in DMF (0.1 mL) was added to2-((dimethylamino)methyl)acrylic acid hydrochloride (14.9 mg, 0.090mmol), followed by a solution of(S)-4-(4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenol hydrochloride(0.025 g, 0.075 mmol) and DIPEA (0.065 ml, 0.375 mmol), the reactionmixture was stirred at rt for 15 minutes and the crude liquid waspurified via preparatory scale C18 column chromatography using agradient of acetonitrile in water with 0.05% trifluoroacetic acid toyield(S)-2-((dimethylamino)methyl)-1-(3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-en-1-oneas a TFA salt (2.6 mg, 0.005 mmol, 7% yield). (m/z): [M+H]⁺ calcd forC₂₃H₂₆N₄O₃ 407.21 found 407.1.

Example 17:(S)-1-(3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-yn-1-one

A solution of HATU (0.031 g, 0.083 mmol) in DMF (0.1 mL) was added topropiolic acid (6.0 mg, 0.090 mmol), followed by a solution of(S)-4-(4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenol hydrochloride(0.025 g, 0.075 mmol) and DIPEA (0.065 ml, 0.375 mmol), the reactionmixture was stirred at rt for 15 minutes and the crude liquid waspurified via preparatory scale C18 column chromatography using agradient of acetonitrile in water with 0.05% trifluoroacetic acid toyield(S)-1-(3-((6-(4-hydroxyphenyl)-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-yn-1-oneas a TFA salt (2.4 mg, 0.005 mmol, 7% yield). (m/z): [M+H]⁺ calcd forC₂₀H₁₇N₃O₃ 348.14 found 348.0.

Example 18:(S)-4-(4-((1-(vinylsulfonyl)pyrrolidin-3-yl)oxy)-1H-indazol-6-yl)phenol

N,N-Diisopropylethylamine (0.052 ml, 0.301 mmol) was added to a solutionof (S)-4-(4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenol hydrochloride(0.020 g, 0.060 mmol) in DMF (0.2 ml) at 0° C., followed byethenesulfonyl chloride (8.4 mL, 0.066 mmol). The reaction mixture wasstirred at rt for 10 minutes and the crude liquid was purified viapreparatory scale C18 column chromatography using a gradient ofacetonitrile in water with 0.05% trifluoroacetic acid to yield(S)-4-(4-((1-(vinylsulfonyl)pyrrolidin-3-yl)oxy)-1H-indazol-6-yl)phenolas a TFA salt (8.2 mg, 0.016 mmol, 27% yield). (m/z): [M+H]⁺ calcd forC₁₉H₁₉N₃O₄ 386.12 found 386.3.

Preparation 52: tert-butyl3-(2-((6-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate

Cesium carbonate (6.33 g, 19.42 mmol) was added to a solution oftert-butyl3-(2-((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(3.20 g, 6.47 mmol) and 3-fluoro-4-hydroxybenzeneboronic acid (1.514 g,9.71 mmol) in 1,4-dioxane (20.71 ml) and water (5.18 ml) and thereaction mixture was degassed with nitrogen for 10 minutes.

Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) (0.793 g,0.971 mmol) was added and the reaction mixture was stirred 110° C. for 2hours. The reaction mixture was concentrated in vacuo to a volume ofabout 5 mL. A saturated aqueous solution of ammonium chloride (20 mL)was added and the mixture was extracted with methylene chloride (2×20mL). The methylene chloride extracts were combined, dried over sodiumsulfate and concentrated in vacuo to yield a brown liquid. The crudeliquid was purified via flash column chromatography using 50% ethylacetate in hexanes to yield tert-butyl3-(2-((6-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(2.30 g, 4.38 mmol, 68% yield). (m/z): [M+H]⁺ calcd for C₂₉H₃₇FN₃O₅526.27 found 526.3.

Preparation 53:4-(4-((2-azetidin-3-yl)propan-2-yl)oxy)-1-H-indazole-6-yl)-2-fluorophenol

Tert-butyl3-(2-((6-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(3.10 g, 5.90 mmol) was dissolved in dichloromethane (10 ml) and TFA (10ml) was slowly added. The clear solution was stirred at rt for 2 hoursupon which LCMS indicated good conversion to the desired product. Thereaction was concentrated down to provide4-(4-((2-azetidin-3-yl)propan-2-yl)oxy)-1-H-indazole-6-yl)-2-fluorophenolas a TFA salt (100% yield). (m/z): [M+H]⁺ calcd for C₁₉H₂₁FN₃O₂ 342.16found 342.3.

Example 19:(E)-1-(3-(2-((6-(3-fluoro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)propan-2-yl)azetidin-1-yl)-4-(dimethylamino)but-2-en-1-one

HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, 2.92 g, 7.07 mmol) was added to a solution of(E)-4-(dimethylamino)but-2-enoic acid hydrochloride (1.07 g, 6.48 mmol)in DMF (10 mL). The reaction mixture was stirred at rt for 10 minutesthen4-(4-((2-azetidin-3-yl)propan-2-yl)oxy)-1-H-indazole-6-yl)-2-fluorophenolTFA salt (2.01 g, 5.89 mmol) was added, followed by DIPEA (3.08 ml, 17.7mmol). The reaction mixture was stirred at rt for 15 minutes and thenconcentrated in vacuo to yield a yellow liquid. The crude liquid waspurified via preparatory scale C18 column chromatography using agradient of 20-80% acetonitrile in water with 0.05% trifluoroacetic acidto yield(E)-4-(dimethylamino)-1-(3-(2-((6-(3-fluoro-4-hydroxyphenyl)-1H-indazol-4-yl)oxy)propan-2-yl)cyclobutyl)but-2-en-1-oneas a TFA salt (1.65 g, 3.65 mmol, 61.9% yield). (m/z): [M+H]⁺ calcd forC₂₅H₃₀FN₄O₃ 453.23 found 453.3.

Preparation 54: tert-butyl3-(2-((2,6-dichloropyridin-4-yl)oxy)propan-2-yl)azetidine-1-arboxylate

A solution of tert-butyl 3-(2-hydroxypropan-2-yl)azetidine-1-carboxylate(6.5 g, 30.21 mmol) in DMSO (10 mL) was added dropwise to a solution of60% by weight sodium hydride in mineral oil (1.65 g, 41.20 mmol) in DMSO(15 mL) and the reaction mixture was stirred at rt for 10 minutes. Asolution of 2,4,6-trichloropyridine (5.0 g, 27.62 mmol) in DMSO (25 mL)was added dropwise to the resulting suspension, and the reaction mixturewas stirred at 60° C. for 16 h. The reaction mixture was quenched usinga saturated aqueous solution of ammonium chloride and extracted withethyl acetate. The ethyl acetate extracts were combined, washed with anaqueous saturated solution of sodium chloride, dried over sodium sulfateand concentrated in vacuo to yield the crude liquid. The crude liquidwas purified via flash column chromatography using 5% ethyl acetate inhexanes to yield tert-butyl3-(2-((2,6-dichloropyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(2.10 g, 5.80 mmol, 21.1% yield). (m/z): [M+H]⁺ calculated forC₁₆H₂₃Cl₂N₂O₃ 361.11 found 361.11.

Preparation 55: tert-butyl3-(2-((6-chloro-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate

A 2M solution of nBuLi in hexanes (3.05 mL, 6.11 mmol) was addeddropwise to a solution of tert-butyl3-(2-((2,6-dichloropyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(2.0 g, 5.55 mmol) in THF (40 mL) at −78° C. DMF (0.64 mL, 8.33 mmol)was added in one portion and the reaction mixture was stirred at −78° C.for 10 min. 5% Acetic acid in diethyl ether was added and the mixturewas further diluted with water. The aqueous layer was extracted withethyl acetate (3 times). The ethyl acetate extracts were combined,washed with an aqueous saturated solution of sodium chloride, dried oversodium sulfate and concentrated in vacuo to yield the crude intermediatetert-butyl3-(2-((2,6-dichloro-3-formylpyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate.Hydrazine hydrate (1.0 mL, 20.56 mmol) was added dropwise to a solutionof the crude tert-butyl3-(2-((2,6-dichloro-3-formylpyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylatein THF (40 mL) at 0° C., and the reaction mixture was stirred at rt for2 h. Triethylamine (1.45 mL, 10.28 mmol) was added, and the reactionmixture was stirred at 60° C. for 16 h. The reaction mixture wasconcentrated in vacuo and the resulting residue was diluted with waterand extracted with ethyl acetate. The ethyl acetate extracts werecombined, washed with an aqueous saturated solution of sodium chloride,dried over sodium sulfate and concentrated in vacuo to yield the crudeliquid. The crude liquid was purified via flash column chromatographyusing 20% ethyl acetate in hexanes to yield tert-butyl3-(2-((6-chloro-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(1.20 g, 3.27 mmol, 58.9% yield). (m/z): [M+H]⁺ calculated forC₁₇H₂₃ClN₄O₃ 367.15 found 367.00.

Preparation 56: tert-butyl3-(2-((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate

Sodium carbonate (636 mg, 6.00 mmol) was added to a solution oftert-butyl3-(2-((6-chloro-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(1.1 g, 3.00 mmol) in 4:1 dioxane/water (15 mL), followed byphenolboronic acid (496 mg, 3.60 mmol) and the reaction mixture wasdegassed with argon for 10 minutes.Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethane adduct (245 mg, 0.3 mmol) was added, the reactionmixture was degassed further with argon for 5 minutes and the reactionmixture was stirred under microwave irradiation at 130° C. for 1 hour.The reaction mixture was diluted with water and extracted with ethylacetate. The ethyl acetate extracts were combined, washed with asaturated aqueous solution of sodium chloride, dried over sodium sulfateand concentrated in vacuo to yield the crude liquid. The crude liquidwas purified via flash column chromatography using 50% ethyl acetate inhexanes to yield tert-butyl3-(2-((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(650 mg, 1.53 mmol, 51.0% yield). (m/z): [M+H]⁺ calcd for C₂₃H₂₉N₄O₄425.22 found 425.21.

Preparation 57:4-(4-((2-(azetidin-3-yl)propan-2-yl)oxy)-1H-pyrazolo[3,4-b]pyridin-6-yl)phenol

Tert-butyl3-(2-((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)propan-2-yl)azetidine-1-carboxylate(630 mg, 1.48 mmol) was dissolved in dichloromethane (10 ml) and TFA(6.3 ml) was slowly added. The clear solution was stirred at rt for 4hours upon which LCMS indicated good conversion to the desired product.The reaction was concentrated in vacuo and the residue was trituratedwith acetonitrile and diethyl ether to yield4-(4-((2-(azetidin-3-yl)propan-2-yl)oxy)-1H-pyrazolo[3,4-b]pyridin-6-yl)phenolas a TFA salt (100% yield). (m/z): [M+H]⁺ calcd for C₁₈H₂₁N₄O₂ 325.17found 325.13.

Example 20:1-(3-(2-((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)propan-2-yl)azetidin-1-yl)prop-2-en-1-one

Diisopropylethylamine (0.298 ml, 1.711 mmol) was added to a solution of4-(4-((2-(azetidin-3-yl)propan-2-yl)oxy)-1H-pyrazolo[3,4-b]pyridin-6-yl)phenolTFA salt (150 mg, 0.342 mmol) in DMF (3.00 ml) at 0° C., followed byacryloyl chloride (0.031 ml, 0.376 mmol) and the reaction mixture wasstirred at rt for 15 min. The reaction mixture was concentrated in vacuoto yield a brown liquid. The crude liquid was purified via preparatoryscale C18 column chromatography using a gradient of 10-70% acetonitrilein water with 0.05% trifluoroacetic acid to yield1-(3-(2-((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)propan-2-yl)azetidin-1-yl)prop-2-en-1-oneas a TFA salt (80.5 mg, 0.163 mmol, 47.8% yield). (m/z): [M+H]⁺ calcdfor C₂₁H₂₃N₄O₃ 379.18 found 379.3.

Preparation 58: tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)-3-methylazetidine-1-carboxylate

Sodium hydride (211 mg, 8.79 mmol) was added to a solution of tert-butyl3-(hydroxymethyl)-3-methylazetidine-1-carboxylate (973 mg, 4.83 mmol) inDMF (4.4 mL) at 0° C. and the reaction mixture was stirred at rt for 30minutes. The reaction mixture was then added dropwise to a solution of4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine(1.20 g, 4.39 mmol) in DMF (4.4 mL) at 0° C. and the reation mixture wasstirred at rt for 2 hours. The reaction was quenched with addition ofwater (1 mL) and the resulting mixture was concentrated in vacuo toyield a light brown liquid. Water (10 mL) was added and the mixture wasextracted with ethyl acetate (3×20 mL). The ethyl acetate extracts werecombined, washed with a saturated aqueous solution of sodium chloride (5mL) and dried over sodium sulfate to yield a brown liquid. The crudeliquid was purified via flash column chromatography using 40% ethylacetate in hexanes to yield tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)-3-methylazetidine-1-carboxylate(1.06 g, 4.39 mmol, 54.9% yield). (m/z): [M+H]⁺ calcd for C₂₀H₂₉ClN₅O₄438.19 found 438.6.

Preparation 59: tert-butyl3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)-3-methylazetidine-1-carboxylate

Potassium phosphate tribasic (218 mg, 1.03 mmol) was added to a solutionof tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)-3-methylazetidine-1-carboxylate(150 mg, 0.343 mmol) in 4:1 dioxane/water (1.71 mL), followed by3-fluoro-4-hydroxyphenylboronic acid (80.0 mg, 0.514 mmol) and thereaction mixture was degassed with nitrogen for 10 minutes. Palladiumacetate (15.4 mg, 0.069 mmol) and 1,1′-bis(di-t-butylphosphino)ferrocene(32.5 mg, 0.069 mmol) were added and the reaction mixture was stirred at110° C. for overnight. The reaction mixture was concentrated in vacuo toa volume of about 5 mL. A saturated aqueous solution of ammoniumchloride (20 mL) was added and the mixture was extracted with ethylacetate (3×20 mL). The ethyl acetate extracts were combined, dried oversodium sulfate and concentrated in vacuo to yield a brown liquid. Thecrude liquid was purified via flash column chromatography using 40%ethyl acetate in hexanes to yield tert-butyl3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)-3-methylazetidine-1-carboxylate(65 mg, 0.343 mmol, 37.0% yield). (m/z): [M+H]⁺ calcd for C₂₆H₃₃FN₅O₅514.25 found 514.7.

Preparation 60:2-fluoro-4-(4-((3-methylazetidin-3-yl)methoxy)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol

Tert-butyl3-(((6-(3-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)-3-methylazetidine-1-carboxylate(65 mg, 0.127 mmol) was dissolved in dichloromethane (0.56 ml) and TFA(0.56 ml) was added and the reaction mixture was stirred at rt for 4hours. The reaction was concentrated in vacuo to yield2-fluoro-4-(4-((3-methylazetidin-3-yl)methoxy)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenolas a TFA salt (100% yield). (m/z): [M+H]⁺ calcd for C₁₆H₁₇FN₅O₂ 330.14found 330.3.

Example 21:1-(3-(((6-(3-fluoro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)-3-methylazetidin-1-yl)prop-2-en-1-one

Diisopropylethylamine (0.210 ml, 1.20 mmol) was added to a solution of2-fluoro-4-(4-((3-methylazetidin-3-yl)methoxy)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenolTFA salt (53.2 mg, 0.12 mmol) in DMF (0.60 ml) at 0° C., followed byacryloyl chloride (9.75 μl, 0.120 mmol) and the reaction mixture wasstirred at rt for 15 min. The reaction mixture was concentrated in vacuoto yield the crude liquid. The crude liquid was purified via preparatoryscale C18 column chromatography using a gradient of 10-60% acetonitrilein water with 0.05% trifluoroacetic acid to yield1-(3-(((6-(3-fluoro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)methyl)-3-methylazetidin-1-yl)prop-2-en-1-oneas a TFA salt (10.5 mg, 0.120 mmol, 21.7% yield). (m/z): [M+H]⁺ calcdfor C₁₉H₁₉FN₅O₃ 384.15 found 384.3.

Preparation 61: tert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)-3-ethylazetidine-1-carboxylate

60% By weight sodium hydride in mineral oil (353 mg, 8.82 mmol) wasadded to a solution of tert-butyl3-ethyl-3-(hydroxymethyl)azetidine-1-carboxylate (1.1 g, 4.85 mmol) inDMF (10 mL) at 0° C. and the reaction mixture was stirred at rt for 40minutes. A solution of4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine(1.20 g, 4.41 mmol) in DMF (5 mL) at 0° C. and the reaction mixture wasstirred at rt for 1 hour. The reaction was quenched with addition ofwater and the mixture was extracted with ethyl acetate. The ethylacetate extracts were combined, washed with water and a saturatedaqueous solution of sodium chloride and then dried over sodium sulfateto yield the crude residue. The crude residue was purified via flashcolumn chromatography using 5% ethyl acetate in hexanes to yieldtert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)-3-ethylazetidine-1-carboxylate(900 mg, 4.39 mmol, 45.2% yield). (m/z): [M+H]⁺ calcd for C₂₂H₃₂ClN₄O₄451.21 found 451.13.

Preparation 62: tert-butyl3-ethyl-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate

A solution of phenolboronic acid (413 mg, 2.99 mmol) and sodiumcarbonate (421 mg 3.98 mmol) in water (2 mL) was added to solution oftert-butyl3-(((6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)-3-ethylazetidine-1-carboxylate(900 mg, 1.99 mmol) in dioxane (8 mL) and the reaction was degassed withargon for 5 minutes.

Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethane adduct (162 mg, 0.19 mmol) was added, the reactionmixture was degassed further with argon for 5 minutes and the reactionmixture was stirred at 110° C. for 1 hour. The reaction mixture wasdiluted with ethyl acetate and filtered through Celite. Water was added,the ethyl acetate layer was separated and the aqueous layer wasextracted with ethyl acetate. The ethyl acetate extracts were combined,washed with a saturated aqueous solution of sodium chloride, dried oversodium sulfate and concentrated in vacuo to yield the crude liquid. Thecrude liquid was purified via flash column chromatography using 30%ethyl acetate in hexanes to yield tert-butyl3-ethyl-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate(800 mg, 1.57 mmol, 79.0% yield). (m/z): [M+H]⁺ calcd for C₂₈H₃₇N₄O₅509.28 found 509.29.

Preparation 63:4-(4-((3-ethylazetidin-3-yl)methoxy)-1H-pyrazolo[3,4-b]pyridin-6-yl)phenol

Tert-butyl3-ethyl-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidine-1-carboxylate(800 mg, 1.57 mmol) was dissolved in dichloromethane (10 ml) and TFA (10ml) was added at 0° C. and the reaction mixture was stirred at rt for 3hours. The reaction was concentrated in vacuo and the resulting residuewas triturated with diethyl ether. The crude residue was dissolved in aminimum of acetonitrile and precipitated with diethyl ether to yield4-(4-((3-ethylazetidin-3-yl)methoxy)-1H-pyrazolo[3,4-b]pyridin-6-yl)phenolas a TFA salt (287 mg, 0.885 mmol, 56.3% yield). (m/z): [M+H]⁺ calcd forC₁₈H₂₁N₄O₂ 325.17 found 325.07.

Example 22:1-(3-ethyl-3-(((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidin-1-yl)prop-2-en-1-one

Diisopropylethylamine (0.246 ml, 1.41 mmol) was added to a solution of4-(4-((3-ethylazetidin-3-yl)methoxy)-1H-pyrazolo[3,4-b]pyridin-6-yl)phenolTFA salt (124 mg, 0.283 mmol) in DMF (2.0 ml) at 0° C., followed byacryloyl chloride (25 μl, 0.311 mmol) and the reaction mixture wasstirred at rt for 15 min. The reaction mixture was concentrated in vacuoto yield a brown liquid. The crude liquid was purified via preparatoryscale C18 column chromatography using a gradient of 20-80% acetonitrilein water with 0.05% trifluoroacetic acid to yield1-(3-ethyl-3-(((6-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)oxy)methyl)azetidin-1-yl)prop-2-en-1-oneas a TFA salt (44.8 mg, 0.118 mmol, 41.9% yield). (m/z): [M+H]⁺ calcdfor C₂₁H₂₃N₄O₃ 379.18 found 379.3.

Preparation 64: 6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazole

A solution of sodium nitrite (1.85 g, 26.8 mmol) in water (50 mL) wasadded to a solution of 5-bromo-3-fluorobenzene-1,2-diamine (5.00 g, 24.4mmol) in water (50 mL) and acetic acid (18.0 mL) and the reactionmixture was stirred at rt for 15 minutes and then at 85° C. for 1 hour.The reaction mixture was concentrated in vacuo a water was added to theresulting residue. The mixture was extracted with ethyl acetate (2×300mL), the ethyl acetate extracts were combined, dried over sodium sulfateand concentrated in vacuo to yield6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazole (5.0 g, 23.1 mmol, 94.0%yield). (m/z): [M−H]⁻ calcd for C₆H₃BrFN₃ 213.94 found 213.94.

Preparation 65:6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazole

p-Toluenesulfonic acid (796 mg, 4.62 mmol) was added to a solution of6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazole (5.0 g, 23.1 mmol) intetrahydrofuran (200 mL), followed by 3,4-dihydro-2H-pyran (7.7 g, 92.6mmol) and the reaction mixture was stirred at rt for 4 hours. Thereaction mixture was concentrated in vacuo and the resulting residue waspurified via flash column chromatography using 10% ethyl acetate inhexanes to yield6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazole(3.8 g, 12.7 mmol, 55.4% yield). (m/z): [M+H]⁺ calcd for C₁₁H₁₂BrFN₃O300.01 found 300.09.

Preparation 66: tert-butyl(3S)-3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate

Cesium carbonate (1.50 g, 4.64 mmol) was added to a solution oftert-butyl (S)-3-(hydroxymethyl)pyrrolidine-1-carboxylate (564 mg, 2.80mmol) in DMSO (15 ml) at 0° C., followed by6-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazole(700 mg, 2.34 mmol) and the reaction mixture was stirred at 80° C. for 3hours. Water was added and the mixture was extracted with ethyl acetate(3×). The ethyl acetate extracts were combined, washed with water and anaqueous solution of sodium chloride, dried over sodium sulfate andconcentrated in vacuo to yield the crude residue. The crude residue waspurified via flash column chromatography using 30% ethyl acetate inhexanes to yield tert-butyl(3S)-3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate(500 mg, 1.04 mmol, 45.6% yield). (m/z): [M+H]⁺ calcd for C₂₁H₃₀BrN₄O₄481.15 found 481.2.

Preparation 67: tert-butyl(3S)-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate

Phenolboronic acid (192 mg, 1.40 mmol) was added to solution oftert-butyl(3S)-3-(((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate(450 mg, 0.93 mmol) in dioxane (10 mL) and water (2 mL), followed bypotassium phosphate (394 mg, 1.86 mmol) and the reaction was degassedwith argon for 15 minutes. 2′-(Dimethylamino)-2-biphenylyl-palladium(II)chloride dinorbomylphosphine complex (52 mg, 0.093 mmol) was added andthe reaction mixture was stirred at 110° C. for 40 minutes undermicrowave irradiation. The reaction mixture was filtered through Celite.Water was added and the mixture was extracted with ethyl acetate (3×).The ethyl acetate extracts were combined, washed with a saturatedaqueous solution of sodium chloride, dried over sodium sulfate andconcentrated in vacuo to yield the crude residue. The crude residue waspurified via flash column chromatography using 30% ethyl acetate inhexanes to yield tert-butyl(3S)-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate(300 mg, 0.607 mmol, 65.0% yield). (m/z): [M+H]⁺ calcd for C₂₇H₃₅N₄O₅495.26 found 495.06.

Preparation 68:(S)-4-(4-(pyrrolidin-3-ylmethoxy)-1H-benzo[d][1,2,3]triazol-6-yl)phenol

4M hydrochloric acid in dioxane (10.0 mL, 40 mmol) was added to asolution of((3S)-3-(((6-(4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-benzo[d][1,2,3]triazol-4-yl)oxy)methyl)pyrrolidine-1-carboxylate(230 mg, 0.465 mmol) in methanol (2. mL) and the reaction mixture wasstirred at rt for 5 hours. The reaction was concentrated in vacuo andthe resulting residue was triturated with diethyl ether to yield(S)-4-(4-(pyrrolidin-3-ylmethoxy)-1H-benzo[d][1,2,3]triazol-6-yl)phenolas a hydrochloride salt (225 mg, 0.649 mmol, 85.0% yield). (m/z): [M+H]⁺calcd for C₁₇H₁₉N₄O₂ 311.15 found 311.06.

Example 23:(S)-1-(3-(((6-(4-hydroxyphenyl)-1H-benzo[d][1,2,3]triazol-4-yl)oxy)methyl)pyrrolidin-1-yl)prop-2-en-1-one

Diisopropylethylamine (0.083 ml, 0.475 mmol) was added to a solution of(S)-4-(4-(pyrrolidin-3-ylmethoxy)-1H-benzo[d][1,2,3]triazol-6-yl)phenolhydrochloride salt (32.9 mg, 0.095 mmol) in DMF (0.475 ml) at 0° C.,followed by acryloyl chloride (7.7 μl, 0.095 mmol) and the reactionmixture was stirred at rt for 15 min. The reaction mixture wasconcentrated in vacuo to yield a brown liquid. The crude liquid waspurified via preparatory scale C18 column chromatography using agradient of 5-75% acetonitrile in water with 0.05% trifluoroacetic acidto yield(S)-1-(3-(((6-(4-hydroxyphenyl)-1H-benzo[d][1,2,3]triazol-4-yl)oxy)methyl)pyrrolidin-1-yl)prop-2-en-1-oneas a TFA salt (2.5 mg, 0.068 mmol, 7.2% yield). (m/z): [M+H]⁺ calcd forC₂₀H₂₁N₄O₃ 365.16 found 365.1.

Preparation 69:6-bromo-4-fluoro-3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole

p-Toluenesulfonic acid (236 mg, 1.24 mmol) was added to a solution of6-bromo-4-fluoro-3-methyl-1H-indazole (2.84 g, 12.4 mmol) indichloromethane (41 mL), followed by 3,4-dihydro-2H-pyran (3.13 g, 37.2mmol) and the reaction mixture was stirred at rt for 3 days. Thereaction mixture filtered through a pad of silica gel and the filtratedwas concentrated in vacuo to yield6-bromo-4-fluoro-3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (3.3g, 10.5 mmol, 85% yield). (m/z): [M+Na]⁺ calcd for C₁₃H₁₄BrFN₂NaO 335.02found 335.4.

Example 24:(S)-1-(3-((6-(3-chloro-4-hydroxyphenyl)-3-methyl-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-en-1-one

60% By weight sodium hydride in mineral oil (319 mg, 7.98 mmol) wasadded to a solution of (S)-1-boc-3-hydroxypyrrolidine (1.3 g, 6.94 mmol)in DMF (16 mL) at 0° C. and the reaction mixture was stirred at rt for20 minutes.6-Bromo-4-fluoro-3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (1.00g, 3.19 mmol) was added and the reaction mixture was stirred at rt forovernight. The reaction was quenched with addition of water and themixture was concentrated in vacuo to yield the crude intermediatetert-butyl(3S)-3-((6-bromo-3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylate.

3-chloro-4-hydroxyphenylboronic acid (0.484 g, 2.81 mmol) and potassiumphosphate tribasic (1.19 g, 5.62 mmol) were added to a solution of thecrude tert-butyl(3S)-3-((6-bromo-3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylatein 2:1 dioxane/water (9.5 mL), and the reaction mixture was degassedwith nitrogen for 10 minutes.(2-Dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)methanesulfonate, XPhos-G3-Palladacycle (95 mg, 0.112 mmol) was addedand the reaction mixture was stirred at 110° C. for 3 hours undermicrowave irradiation. Water was added and the mixture was extractedwith ethyl acetate. The ethyl acetate extracts were combined andconcentrated in vacuo to yield the crude intermediate tert-butyl(3S)-3-((6-(3-chloro-4-hydroxyphenyl)-3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylate.

4.0N HCl in dioxane (3.75 ml, 15.0 mmol) was added to a solution of thecrude tert-butyl(3S)-3-((6-(3-chloro-4-hydroxyphenyl)-3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)oxy)pyrrolidine-1-carboxylatein methanol (0.5 mL), and the reaction mixture was stirred at 60° C. for30 minutes. The reaction mixture was concentrated in vacuo to yield thecrude intermediate(S)-2-chloro-4-(3-methyl-4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenol.

N,N-Diisopropylethylamine (4.59 ml, 26.3 mmol) was added to a solutionof the crude intermediate(S)-2-chloro-4-(3-methyl-4-(pyrrolidin-3-yloxy)-1H-indazol-6-yl)phenolin methylene chloride (3.75 ml), followed by acryloyl chloride (60.9 μL,0.750 mmol). The reaction mixture was stirred at rt for 30 minutes thenconcentrated in vacuo to yield the crude residue. The crude residue waspurified via preparatory scale C₁₈ column chromatography using agradient of 0-40% acetonitrile in water with 0.05% trifluoroacetic acidto yield(S)-1-(3-((6-(3-chloro-4-hydroxyphenyl)-3-methyl-1H-indazol-4-yl)oxy)pyrrolidin-1-yl)prop-2-en-1-oneas a TFA salt (7.6 mg, 0.015 mmol, 0.98% yield). (m/z): [M+H]⁺ calcd forC₂₁H₂₁ClN₃O₃ 398.13 found 398.3.

The compounds of Tables 1-12 were prepared using similar syntheticmethods.

TABLE 1

Ex Calc Found No. R¹ R² R³ R⁴ R⁵ R⁶ X¹ X² R^(k1) R^(k2) Formula [M + H]⁺[M + H]⁺ 1-1

H H H H H CH CH H H C₂₃H₂₆N₄O₃ 407.20 407 1-2

H H H H H CH CH H H C₂₀H₁₉N₃O₃ 350.13 350 1-3

Cl H H H H CH CH H H C₂₃H₂₅ClN₄O₃ 441.15 441.1 1-4

F H H H H CH CH H H C₂₃H₂₅FN₄O₃ 425.18 425.1 1-5

F H H H H CH CH H H C₂₀H₁₈FN₃O₃ 368.12 368 1-6

H H H H H N N H H C₁₈H₁₇N₅O₃ 352.12 352.3 1-7

H H H H H CH N H H C₂₂H₂₅N₅O₃ 408.18 408.2 1-8

H H H H H CH N H H C₁₉H₁₈N₄O₃ 351.12 351.2 1-9

H H H H H N CH H H C₁₉H₁₈N₄O₃ 351.12 351.1 1-10

H H H H H CH CH Me H C₂₄H₂₈N₄O₃ 421.20 421.3 1-11

H H H H H CH CH Me H C₂₁H₂₁N₃O₃ 364.14 364.2 1-12

H H H H H CH CH H Me C₂₁H₂₁N₃O₃ 364.14 364 1-13

F F H H H CH CH H H C₂₀H₁₇F₂N₃O₄ 386.11 386 1-14

F H F H H CH CH H H C₂₀H₁₇F₂N₃O₃ 386.11 386 1-15

F Cl H H H CH CH H H C₂₀H₁₇ClFN₇O₃ 402.08 402 1-16

F H H Me H CH CH H H C₂₁H₂₀FN₃O₃ 382.13 382.1 1-17

H F H H H CH CH H H C₂₀H₁₈FN₃O₃ 368.12 368.0 1-18

H F H F H CH CH H H C₂₀H₁₇F₂N₃O₃ 386.11 386.0 1-19

F H H F H CH CH H H C₂₀H₁₇F₂N₃O₃ 386.11 386.0 1-20

H H H H Me CH CH H H C₂₁H₂₁N₃O₃ 364.14 364.0 1-21

H H H H NHMe CH CH H H C₂₁H₂₂N₄O₃ 379.15 379.0 1-22

H H H H CH₂—OMe CH CH H H C₂₂H₂₃N₃O₄ 394.15 394.0 1-23

F H H H Me CH CH H H C₂₁H₂₀FN₃O₃ 382.13 382.0 1-24

H H H H H N N Me H C₁₉H₁₉N₅O₃ 366.13 366.0 1-25

H H H H CHF₂ N N H H C₁₉H₁₇F₂N₅O₃ 402.11 402.0 1-26

H H H H H N CH Me H C₂₀H₂₀N₄O₃ 365.14 365.0 1-27

F H H H H CH N H H C₁₉H₁₇FN₄O₃ 369.11 369.2 1-28

F H H H H CH N H H C₂₂H₂₄FN₅O₃ 426.17 426.1 1-29

F H H Me H CH CH H H C₂₄H₂₇FN₄O₃ 439.19 439.1 1-30

F H H F Me CH CH H H C₂₁H₁₉F₂N₃O₃ 400.12 400.4 1-31

H H H H Me N CH H H C₂₀H₂₀N₄O₃ 365.14 365.0 1-32

Cl H H H Me N CH H H C₂₀H₁₉ClN₄O₃ 399.10 399.0 1-33

F H H H Me N CH H H C₂₀H₁₉FN₄O₃ 383.13 383.0 1-34

H H H H Me CH N H H C₂₀H₂₀N₄O₃ 365.14 365.0 1-35

Cl H H H Me CH N H H C₂₀H₁₉ClN₄O₃ 399.10 399.0 1-36

F H H H Me CH N H H C₂₀H₁₉FN₄O₃ 383.13 383.0 1-37

Cl H H H Me N N H H C₁₉H₁₈ClN₅O₃ 400.09 400.0 1-38

F H H H Me N N H H C₁₉H₁₈FN₅O₃ 384.12 384.0 1-39

H H H H SMe CH CH H H C₂₁H₂₁N₃O₃S 396.11 396.4 1-40

F H H H SMe CH CH H H C₂₁H₂₀FN₃O₃S 414.11 414.0 1-41

Cl H H H SMe CH CH H H C₂₁H₂₀ClN₃O₃S 430.08 430.0 1-42

H H H H NMe₂ CH CH H H C₂₂H₂₄N₄O₃ 393.17 393.0 1-43

H H H H CF₃ CH CH H H C₂₁H₁₈F₃N₃O₃ 418.11 418.4 1-44

H H H H OMe CH CH H H C₂₁H₂₁N₃O₄ 380.14 380.0 1-45

H H H H CN CH CH H H C₂₁H₁₈N₄O₃ 375.12 375.0 1-46

Cl H H H OMe CH CH H H C₂₁H₁₇ClN₄O₃ 409.08 409.0 1-47

H H H H Cl CH CH H H C₂₀H₁₈ClN₃O₃ 384.09 384.1 1-48

H H H H Cl CH CH H H C₂₃H₂₅ClN₄O₃ 441.15 441.8

TABLE 2

Calc Found Ex No. R¹ R² R³ X¹ X² Formula [M + H]⁺ [M + H]⁺ 2-1

H H CH CH C₂₄H₂₈N₄O₃ 421.20 421.2 2-2

Cl H CH CH C₂₄H₂₇N₄O₃Cl 455.16 455.1 2-3

Cl H CH CH C₂₁H₂₀ClN₃O₃ 398.10 398.2 2-4

F H CH CH C₂₄H₂₇FN₄O₃ 439.19 439.3 2-5

F H CH CH C₂₁H₂₀FN₃O₃ 382.13 382.1 2-6

H H N CH C₂₀H₂₀N₄O₃ 365.14 365.0 2-7

H H N N C₁₉H₁₉N₅O₃ 366.13 366.0 2-8

H H CH N C₂₀H₂₀N₄O₃ 365.14 365.0 2-9

Cl H CH N C₂₃H₂₆ClN₅O₃ 456.16 456.2 2-10

F H CH N C₂₀H₁₉FN₄O₃ 383.13 383.0 2-11

Cl H N N C₁₉H₁₈ClN₅O₃ 400.09 400.1 2-12

H H N CH C₂₀H₂₀N₄O₃ 365.14 365.0 2-13

F H CH N C₂₃H₂₆FN₅O₃ 440.19 440.3 2-14

Cl H CH N C₂₀H₁₉ClN₄O₃ 399.10 399.0 2-15

F H N N C₁₉H₁₈FN₅O₃ 384.12 384.1 2-16

Cl H N CH C₂₀H₁₉ClN₄O₃ 399.10 399.1 2-17

F H N CH C₂₀H₁₉FN₄O₃ 383.13 383.1

TABLE 3

Ex Calc Found No. R¹ R² R³ X¹ X² R^(k1) R^(k2) R^(d) R^(e) Formula [M +H]⁺ [M + H]⁺ 3-1

H H CH CH H H H H C₂₀H₁₉N₃O₃ 350.13 350.2 3-2

Cl H CH CH H H H H C₂₃H₂₅ClN₄O₃ 441.15 441.3 3-3

Cl H CH CH H H H H C₂₀H₁₈ClN₃O₃ 384.09 384.1 3-4

F H CH CH H H H H C₂₀H₁₈FN₃O₃ 368.12 368.2 3-5

H H CH CH F H H H C₂₃H₂₅FN₄O₃ 425.18 425.1 3-6

H H CH CH F H H H C₂₀H₁₈FN₃O₃ 368.12 368.2 3-7

H H CH CH Me H H H C₂₄H₂₈N₄O₃ 421.20 421.1 3-8

H H CH CH Me H H H C₂₁H₂₁N₃O₃ 364.14 364.1 3-9

Cl H CH CH Me H H H C₂₄H₂₇ClN₄O₃ 455.16 456.1 3-10

Cl H CH CH Me H H H C₂₁H₂₀ClN₃O₃ 398.10 398.1 3-11

F H CH CH Me H H H C₂₄H₂₇FN₄O₃ 439.19 439.2 3-12

F H CH CH Me H H H C₂₁H₂₀FN₃O₃ 382.13 382.1 3-13

H H CH CH Et H H H C₂₅H₃₀N₄O₃ 435.22 435.1 3-14

H H CH CH Et H H H C₂₂H₂₃N₃O₃ 378.16 378.1 3-15

Cl H CH CH Et H H H C₂₅H₂₉ClN₄O₃ 469.18 469.1 3-16

Cl H CH CH Et H H H C₂₂H₂₂ClN₃O₃ 412.12 412.1 3-17

F H CH CH Et H H H C₂₅H₂₉FN₄O₃ 453.21 453.3 3-18

F H CH CH Et H H H C₂₂H₂₂FN₃O₃ 396.15 396.1 3-19

H H CH CH CN H H H C₂₄H₂₅N₅O₃ 432.18 432.1 3-20

H H CH CH CN H H H C₂₁H₁₈N₄O₃ 375.12 375.1 3-21

Cl H CH CH CN H H H C₂₄H₂₄ClN₅O₃ 466.14 466.2 3-22

Cl H CH CH CN H H H C₂₁H₁₇ClN₄O₃ 409.08 409.1 3-23

F H CH CH CN H H H C₂₁H₁₇FN₄O₃ 393.11 393.1 3-24

H H CH CH H H Me Me C₂₅H₃₀N₄O₃ 435.22 435.0 3-25

H H CH CH H H Me Me C₂₂H₂₃N₃O₃ 378.16 378.1 3-26

Cl H CH CH H H Me Me C₂₂H₂₂ClN₃O₃ 412.12 412.2 3-27

F H CH CH H H Me Me C₂₅H₂₉FN₄O₃ 453.21 453.2 3-28

F H CH CH H H Me Me C₂₂H₂₂FN₃O₃ 393.15 396.3 3-29

H H CH N H H H H C₁₉H₁₈N₄O₃ 351.12 351.0 3-30

H H CH CH H Me H H C₂₄H₂₈N₄O₃ 421.20 421.2 3-31

H H N N H Me H H C₁₉H₁₉N₅O₃ 366.13 366.1 3-32

H H CH CH OMe H H H C₂₄H₂₈N₄O₄ 437.20 437.3 3-33

H H CH CH OMe H H H C₂₁H₂₁N₃O₄ 380.14 380.2 3-34

Cl H CH N H H H H C₂₂H₂₄ClN₅O₃ 442.14 442.1 3-35

Cl H CH N H H H H C₁₉H₁₇ClN₄O₃ 385.08 385.0 3-36

F H CH N H H H H C₂₂H₂₄FN₅O₃ 426.17 426.0 3-37

F H CH N H H H H C₁₉H₁₇FN₄O₃ 369.11 369.1 3-38

Cl H N N H H H H C₁₈H₁₆ClN₅O₃ 386.08 386.2 3-39

F H N N H H H H C₁₈H₁₆FN₅O₃ 370.11 370.1 3-40

F H CH CH F H H H C₂₃H₂₄F₂N₄O₃ 443.17 443.1 3-41

F H CH CH F H H H C₂₀H₁₇F₂N₃O₃ 386.11 386.2 3-42

Cl H CH CH F H H H C₂₃H₂₄ClFN₄O₃ 459.14 459.1 3-43

Cl H CH CH F H H H C₂₀H₁₇ClFN₃O₃ 402.08 402.0 3-44

H H CH N F H H H C₂₂H₂₄FN₅O₃ 426.17 426.2 3-45

H H N N F H H H C₁₈H₁₆FN₅O₃ 370.11 370.1 3-46

H H N CH F H H H C₁₉H₁₇FN₄O₃ 369.11 369.3 3-47

Cl H CH N F H H H C₁₉H₁₆ClFN₄O₃ 403.07 403.0 3-48

F H CH N F H H H C₂₂H₂₃F₂N₅O₃ 444.16 444.0 3-49

F H CH N F H H H C₁₉H₁₆F₂N₄O₃ 387.10 387.0 3-50

Cl H N N F H H H C₂₁H₂₂ClFN₆O₃ 461.13 461.1 3-51

F H N N F H H H C₂₁H₂₂F₂N₆O₃ 445.16 445.2 3-52

Cl H N CH F H H H C₂₂H₂₃ClFN₅O₃ 460.13 460.1 3-53

F H N CH F H H H C₂₂H₂₃F₂N₅O₃ 444.16 444.2 3-54

F H N CH F H H H C₁₉H₁₆F₂N₄O₃ 387.10 387.1 3-55

H H CH N Me H H H C₂₃H₂₇N₅O₃ 422.20 422.2 3-56

H H CH N Me H H H C₂₀H₂₀N₄O₃ 365.14 365.2 3-57

H H N N Me H H H C₁₉H₁₉N₅O₃ 366.13 366.1 3-58

H H N CH Me H H H C₂₀H₂₀N₄O₃ 365.14 365.2 3-59

F H CH N Me H H H C₂₀H₁₉FN₄O₃ 383.13 383.3 3-60

Cl H N N H H H H C₂₁H₂₃ClN₆O₃ 443.14 443.1 3-61

H H CH N Et H H H C₂₄H₂₉N₅O₃ 436.21 436.1 3-62

H H CH N Et H H H C₂₁H₂₂N₄O₃ 379.15 379.0 3-63

H H N N Et H H H C₂₀H₂₁N₅O₃ 380.15 380.2 3-64

H H CH N Et H H H C₂₄H₂₉N₅O₃ 436.21 436.1 3-65

H H CH N Et H H H C₂₁H₂₂N₄O₃ 379.15 379.0 3-66

Cl H CH N Et H H H C₂₁H₂₁ClN₄O₃ 413.11 413.1 3-67

F H N N Et H H H C₂₀H₂₀FN₅O₃ 398.14 398.1 3-68

Cl H N CH Et H H H C₂₄H₂₈ClN₅O₃ 470.17 470.2 3-69

Cl H N CH Et H H H C₂₁H₂₁ClN₄O₃ 413.11 413.2 3-70

F H N CH Et H H H C₂₁H₂₁FN₄O₃ 397.14 397.2 3-71

Cl H CH N CN H H H C₂₃H₂₃ClN₆O₃ 467.14 467.0 3-72

Cl H CH CH OMe H H H C₂₄H₂₇ClN₄O₄ 471.16 471.2 3-73

Cl H CH CH OMe H H H C₂₁H₂₀ClN₃O₄ 414.10 414.2 3-74

F H CH CH OMe H H H C₂₄H₂₇FN₄O₄ 455.19 455.1 3-75

F H CH CH OMe H H H C₂₁H₂₀FN₃O₄ 398.13 398.2 3-76

H H N N OMe H H H C₁₉H₁₉N₅O₄ 382.13 382.1 3-77

H H N N H H H Me C₁₉H₁₉N₅O₃ 366.13 366.1 3-78

H H N N H H H Me (S) C₁₉H₁₉N₅O₃ 366.13 366.1 3-79

H H N CH H H H H C₂₂H₂₅N₅O₃ 408.18 408.1 3-80

Cl H N CH H H Me Me C₂₁H₂₁ClN₄O₃ 413.11 413.2 3-81

F H N CH H H Me Me C₂₁H₂₁FN₄O₃ 397.14 397.1 3-82

F F CH CH H H H H C₂₃H₂₄F₂N₄O₃ 443.17 443.7 3-83

OMe H CH CH H H H H C₂₁H₂₁N₃O₄ 380.14 380.0 3-84

H H CH CH H H H H C₂₀H₁₇N₃O₃ 348.11 348.1 3-85

H H CH CH H H H H C₂₅H₂₈N₄O₃ 433.20 433.1 3-86

Cl H N CH H H H H C₁₉H₁₇ClN₄O₃ 385.08 385.1 3-87

F H N CH H H H H C₁₉H₁₇FN₄O₃ 369.11 369.1 3-88

H H N N F H H H C₂₁H₂₃FN₆O₃ 427.17 427.2 3-89

H H N CH F H H H C₂₂H₂₄FN₅O₃ 426.17 426.2 3-90

H H CH N F H H H C₁₉H₁₇FN₄O₃ 369.11 369.1 3-91

Cl H CH N F H H H C₂₂H₂₃ClFN₅O₃ 460.13 460.0 3-92

Cl H N N F H H H C₁₈H₁₅ClFN₅O₃ 404.07 404.1 3-93

F H N N F H H H C₁₈H₁₅F₂N₅O₃ 388.10 388.1 3-94

Cl H N CH F H H H C₁₉H₁₆ClFN₄O₃ 403.07 403.1 3-95

Cl H CH N Me H H H C₂₃H₂₆ClN₅O₃ 456.16 456.2 3-96

F H CH N Me H H H C₂₃H₂₆FN₅O₃ 440.19 440.2 3-97

Cl H CH N Me H H H C₂₀H₁₉ClN₄O₃ 399.10 399.2 3-98

F H N CH Me H H H C₂₀H₁₉FN₄O₃ 383.13 383.2 3-99

Cl H CH N Et H H H C₂₄H₂₈ClN₅O₃ 470.17 470.1 3-100

F H CH N Et H H H C₂₄H₂₈FN₅O₃ 454.20 454.2 3-101

F H CH N Et H H H C₂₁H₂₁FN₄O₃ 397.14 397.2 3-102

H H CH N CN H H H C₂₃H₂₄N₆O₃ 433.18 433.2 3-103

H H CH N CN H H H C₂₀H₁₇N₅O₃ 376.12 376.2 3-104

H H N N CN H H H C₁₉H₁₆N₆O₃ 377.11 377.0 3-105

H H CH N CN H H H C₂₀H₁₇N₅O₃ 376.12 376.2 3-106

F H CH N CN H H H C₂₃H₂₃FN₆O₃ 451.17 451.1 3-107

Cl H CH N CN H H H C₂₀H₁₆ClN₅O₃ 410.08 410.1 3-108

F H CH N CN H H H C₂₀H₁₆FN₅O₃ 394.11 394.2 3-109

Cl H N N CN H H H C₁₉H₁₅ClN₆O₃ 411.07 411.1 3-110

F H N N CN H H H C₁₉H₁₅FN₆O₃ 395.10 395.1 3-111

F H N CH CN H H H C₂₀H₁₆FN₅O₃ 394.11 394.1 3-112

H H N N H H H H C₂₁H₂₄N₆O₃ 409.18 409.1 3-113

H H CH CH H H H H C₂₅H₃₀N₄O₃ 435.22 435.1 3-114

H H CH CH H Me H H C₂₁H₂₁N₃O₃ 364.14 364.1 3-115

H H CH CH H H H H C₂₅H₂₈N₄O₃ 433.20 433.1 3-116

H H CH CH H H H H C₂₆H₃₂N₄O₃ 449.23 449.2 3-117

H H CH CH H H H H C₂₆H₃₀N₄O₃ 447.22 3-118

H H CH CH H H H H C₂₆H₃₂N₄O₃ 449.23 449.2 3-119

H H CH CH H H H H C₂₅H₃₀N₄O₄ 451.21 451.1 3-120

H H CH CH H H H H C₂₇H₃₄N₄O₅ 495.24 3-121

H H CH CH H H H H C₂₆H₃₂N₄O₅ 481.22 3-122

Cl H CH CH H H H H C₂₃H₂₅ClN₄O₃ 441.15 441.1 3-123

H H CH CH H H H H C₂₄H₂₆N₄O₃ 419.18 419.1 3-124

H H CH CH H H H H C₂₅H₂₈N₄O₃ 433.20 3-125

H H CH CH H H H H C₂₆H₃₀N₄O₃S 479.19 479.2 3-126

H H CH CH H H H H C₂₅H₃₀N₄O₃S 467.18 3-127

H H CH CH H H H H C₂₇H₃₂N₄O₄ 477.23 3-128

H H CH CH H H H H C₂₇H₃₂N₄O₄ 477.23 477.2 3-129

H H CH CH H H H H C₂₅H₂₈N₄O₄ 449.20 449.1 3-130

H H CH CH H H H H C₂₅H₂₈N₄O₃S 465.17 465.1 3-131

H H CH CH H H H H C₂₇H₃₂N₄O₃S 493.20 493.2 3-132

H H CH CH H H H H C₂₅H₂₈N₄O₄ 449.20 3-133

H H CH CH H H H H C₂₆H₃₀N₄O₄ 463.21 463.2 3-134

H H CH CH H H H H C₂₇H₃₂N₄O₄ 477.23

TABLE 4

Calc Found Ex No. R¹ R² R³ R^(b) R^(k) X¹ X² Formula [M + H]⁺ [M + H]⁺4-1

H H Me H CH CH C₂₁H₂₂N₄O₂ 363.15 363.1 4-2

H H H Me CH CH C₂₁H₂₂N₄O₂ 363.15 363 4-3

H H H H CH CH C₂₀H₂₀N₄O₂ 349.14 349.1 4-4

Cl H Me H CH CH C₂₄H₂₈ClN₅O₂ 454.18 454.1 4-5

Cl H Me H CH CH C₂₁H₂₁ClN₄O₂ 397.12 397.0 4-6

F H Me H CH CH C₂₄H₂₈FN₅O₂ 438.21 438.1 4-7

F H Me H CH CH C₂₁H₂₁FN₄O₂ 481.15 381.0 4-8

H H Me H CH N C₂₃H₂₈N₆O₂ 421.21 421.2

TABLE 5

Calc Found Ex No. R¹ R² R³ X¹ X² R^(k) L Formula [M + H]⁺ [M + H]⁺ 5-1

H H CH CH H O C₂₂H₂₄N₄O₃ 393.17 393.4 5-2

H H CH CH H O C₁₉H₁₇N₃O₃ 336.11 336.2 5-3

Cl H CH CH H O C₂₂H₂₃ClN₄O₃ 427.13 427.1 5-4

Cl H CH CH H O C₁₉H₁₆ClN₃O₃ 370.07 370.1 5-5

F H CH CH H O C₂₂H₂₃FN₄O₃ 411.16 411.3 5-6

F H CH CH H O C₁₉H₁₆FN₃O₃ 354.10 354.2 5-7

H H CH CH H NH C₁₉H₁₈N₄O₂ 335.12 335.2 5-8

H H CH CH H NMe C₂₃H₂₇N₅O₂ 406.20 406.1 5-9

H H CH CH H NMe C₂₀H₂₀N₄O₂ 349.14 349.1 5-10

H H CH CH CHF₂ O C₂₃H₂₄F₂N₄O₃ 443.17 443.1 5-11

H H CH CH Me O C₂₃H₂₆N₄O₃ 407.18 407.1 5-12

H H CH CH Me O C₂₀H₁₉N₃O₃ 350.13 350.1 5-13

H H CH CH

O C₂₅H₂₈N₄O₃ 433.20 433.2 5-14

H H CH CH CHF₂ O C₂₀H₁₇F₂N₃O₃ 386.11 386.0 5-15

H H N CH H O C₁₈H₁₆N₄O₃ 337.11 337.1 5-16

Cl H CH CH CHF₂ O C₂₃H₂₃ClF₂N₄O₃ 477.13 477.1 5-17

F H CH CH CHF₂ O C₂₃H₂₃F₃N₄O₃ 461.16 461.2 5-18

Cl H CH CH Me O C₂₃H₂₅ClN₄O₃ 441.15 441.2 5-19

F H CH CH Me O C₂₃H₂₅FN₄O₃ 425.18 425.3 5-20

H H N N H O C₁₇H₁₅N₅O₃ 338.10 338.2 5-21

Cl H CH CH CHF₂ O C₂₀H₁₆ClF₂N₃O₃ 420.07 420.0 5-22

F H CH CH CHF₂ O C₂₀H₁₆F₃N₃O₃ 404.10 404.1 5-23

Cl H CH CH Me O C₂₀H₁₈ClN₃O₃ 384.09 384.2 5-24

F H CH CH Me O C₂₀H₁₈FN₃O₃ 368.12 368.1 5-25

H H CH CH CF₃ O C₂₃H₂₃F₃N₄O₃ 461.16 461.2 5-26

H H CH CH CF₃ O C₂₀H₁₆F₃N₃O₃ 404.10 404.1

TABLE 6

Calc Found Ex No. R¹ R² R³ X¹ X² R^(k1) R^(k2) R^(k3) Formula [M + H]⁺[M + H]⁺ 6-1

H H CH CH H H H C₂₄H₂₈N₄O₃ 421.20 421.2 6-2

H H CH CH H H H C₂₁H₂₁N₃O₃ 364.14 364.2 6-3

H H CH CH Me H H C₂₂H₂₃N₃O₃ 378.16 378.1 6-4

Cl H CH CH Me H H C₂₅H₂₉ClN₄O₃ 469.18 469.2 6-5

Cl H CH CH Me H H C₂₂H₂₂ClN₃O₃ 412.12 412.2 6-6

F H CH CH Me H H C₂₅H₂₉FN₄O₃ 453.21 453.2 6-7

F H CH CH Me H H C₂₂H₂₂FN₃O₃ 396.15 396.1 6-8

H H CH CH H F F C₂₁H₁₉F₂N₃O₃ 400.12 400.0 6-9

H H CH CH Me H H C₂₅H₃₀N₄O₃ 435.22 435.2 6-10

H H CH CH Me H H C₂₂H₂₃N₃O₃ 378.16 378.1 6-11

H H N CH H H H C₂₀H₂₀N₄O₃ 365.14 365.0 6-12

H H N N H H H C₁₉H₁₉N₅O₃ 366.13 366.1 6-13

Cl H CH CH H H H C₂₁H₂₀ClN₃O₃ 398.10 398.1 6-14

F H CH CH H H H C₂₁H₂₀FN₃O₃ 382.13 382.1 6-15

H H CH N H H H C₂₁H₂₀N₄O₃ 365.14 365.1 6-16

F H CH N H H H C₂₀H₁₉FN₄O₃ 383.13 383.0 6-17

Cl H N CH H H H C₂₀H₁₉ClN₄O₃ 399.10 399.3 6-18

F H N CH H H H C₂₀H₁₉FN₄O₃ 383.13 383.0 6-19

Cl H CH CH H H H C₂₄H₂₇ClN₄O₃ 455.16 455.2 6-20

F H CH CH H H H C₂₄H₂₇FN₄O₃ 439.19 439.1 6-21

Cl H CH N H H H C₂₀H₁₉ClN₄O₃ 399.10 399.1 6-22

Cl H N N H H H C₁₉H₁₈ClN₅O₃ 400.09 400.1 6-23

F H N N H H H C₁₉H₁₈FN₅O₃ 384.12 384.0

TABLE 7

Calc Found Ex No. R¹ R² R³ X³ X² R^(k1) R^(k2) R^(k3) R^(b) Formula [M +H]⁺ [M + H]⁺ 7-1

H H CH CH Me H H H C₂₅H₃₁N₅O₂ 434.22 434.2 7-2

H H CH CH Me H H H C₂₂H₂₄N₄O₂ 377.17 377.3 7-3

H H CH CH H H H Me C₂₂H₂₄N₄O₂ 377.17 377 7-4

H H CH CH H H H H C₂₁H₂₂N₄O₂ 363.15 363 7-5

H H CH CH H F F H C₂₄H₂₇F₂N₅O₂ 456.19 456.2 7-6

H H CH CH H F F H C₂₁H₂₀F₂N₄O₂ 399.13 399.2 7-7

Cl H CH CH H H H Me C₂₂H₂₃ClN₄O₂ 411.14 411.2 7-8

Cl H CH CH Me H H H C₂₅H₃₀ClN₅O₂ 468.19 468.2 7-9

F H CH CH Me H H H C₂₂H₂₃FN₄O₂ 395.16 395.1 7-10

F H CH CH H H H Me C₂₂H₂₃FN₄O₂ 395.16 395.3 7-11

Cl H CH CH Me H H H C₂₂H₂₃ClN₄O₂ 411.13 411.2 7-12

F H CH CH Me H H H C₂₅H₃₀FN₅O₂ 452.21 452.2 7-13

H H CH N Me H H H C₂₁H₂₃N₅O₂ 378.16 378.2 7-14

Cl H CH N Me H H H C₂₁H₂₂ClN₅O₂ 412.12 412.2

TABLE 8

Calc Found Ex No. R¹ R² R³ X¹ X² Formula [M + H]⁺ [M + H]⁺ 8-1

H H CH CH C₂₁H₂₁N₃O₃ 364.14 364 8-2

Cl H CH CH C₂₄H₂₇ClN₄O₃ 455.16 455.1 8-3

Cl H CH CH C₂₁H₂₀ClN₃O₃ 398.10 398.0 8-4

F H CH CH C₂₄H₂₇FN₄O₃ 439.19 439.2 8-5

F H CH CH C₂₁H₂₀FN₃O₃ 382.13 382.0 8-6

H H N CH C₂₀H₂₀FN₄O₃ 365.14 365.1 8-7

H H CH N C₂₀H₂₀N₄O₃ 365.14 365.1 8-8

F H N CH C₂₀H₁₉FN₄O₃ 383.13 383.0 8-9

F H CH N C₂₃H₂₆FN₅O₃ 440.19 440.2 8-10

Cl H CH N C₂₀H₁₉ClN₄O₃ 399.10 399.1 8-11

F H CH N C₂₀H₁₉FN₄O₃ 383.13 383.1 8-12

Cl H N N C₁₉H₁₈ClN₅O₃ 400.09 400.2 8-13

F H N N C₁₉H₁₈FN₅O₃ 384.12 384.0 8-14

Cl H N CH C₂₀H₁₉ClN₄O₃ 399.10 399.0

TABLE 9

Calc Found Ex No. R¹ R² R³ X¹ X² L Formula [M + H]⁺ [M + H]⁺ 9-1

H H CH CH O C₂₃H₂₃N₃O₃ 390.16 390.1 9-2

Cl H CH CH O C₂₃H₂₂ClN₃O₃ 424.12 424.0 9-3

Cl H CH CH O C₂₆H₂₉ClN₄O₃ 481.18 481.1 9-4

F H CH CH O C₂₃H₂₂FN₃O₃ 408.15 408.1 9-5

F H CH CH O C₂₆H₂₉FN₄O₃ 465.21 465.1 9-6

H H CH N O C₂₂H₂₂N₄O₃ 391.15 391.1 9-7

Cl H CH N O C₂₂H₂₁ClN₄O₃ 425.11 425.0 9-8

Cl H CH N O C₂₅H₂₈ClN₅O₃ 482.17 482.1 9-9

H H CH CH NH C₂₃H₂₄N₄O₂ 389.17 389.2 9-10

F H CH N O C₂₂H₂₁FN₄O₃ 409.14 409.0 9-11

H H N N O C₂₂H₂₁N₅O₃ 392.15 392.2

TABLE 10

Ex Calc Found No. R¹ R² R³ X¹ X² L Formula [M + H]⁺ [M + H]⁺ 10-1

H H CH CH O C₂₆H₃₀N₄O₃ 447.22 447.2 10-2

H H CH CH O C₂₃H₂₃N₃O₃ 390.16 390.2 10-3

Cl H CH CH O C₂₆H₂₉ClN₄O₃ 481.18 481.3 10-4

Cl H CH CH O C₂₃H₂₂ClN₃O₃ 424.12 424.2 10-5

F H CH CH O C₂₆H₂₉FN₄O₃ 465.21 465.2 10-6

F H CH CH O C₂₃H₂₂FN₃O₃ 408.15 408.1 10-7

H H CH CH NH C₂₃H₂₄N₄O₂ 389.17 389.3

TABLE 11

Ex Calc Found No. R¹ R² R³ X¹ X² Formula [M + H]⁺ [M + H]⁺ 11-1

H H CH CH C₂₁H₂₀N₄O₄ 393.12 393.1 11-2

H H CH CH C₂₄H₂₇N₅O₄ 450.18 450.2

TABLE 12

Calc Found Ex No. R R¹ Formula [M + H]⁺ [M + H]⁺ 12-1

C₂₃H₂₆N₄O₂S 423.16 423.1 12-2

C₂₀H₁₉N₃O₂S 366.10 366.2 12-3

C₂₃H₂₆N₄O₂S 423.16 423.2 12-4

C₂₁H₂₁N₃O₂S 380.12 380.3 12-5

C₂₄H₂₈N₄O₂S 437.18 437.2 12-6

C₂₁H₂₁N₃O₂S 380.12 380.2 12-7

C₂₄H₂₈N₄O₂S 437.18 437.2 12-8

C₂₁H₂₁N₃O₄S 412.11 412.1 12-9

C₂₁H₂₁N₃O₄S 412.11 412.1 12-10 (racemic)

C₂₁H₂₁N₃O₂ 348.15 348.2 12-11 (racemic)

C₂₄H₂₈N₄O₂ 405.21 405.2 12-12 (racemic)

C₂₂H₂₃N₃O₂ 362.16 362.3 12-13 (racemic)

C₂₅H₃₀N₄O₂ 419.22 419.2 12-14

C₂₅H₃₀N₄O₂ 149.22 419.2 12-15

C₂₂H₂₄N₄O₂ 377.17 377   12-16

C₂₂H₂₄N₄O₂ 377.17 377.2 12-17

C₂₂H₂₄N₄O₂ 377.17 377.3 12-18

C₂₀H₂₀N₄O₂ 349.14 349.2 12-19

C₂₄H₂₈N₄O₃ 421.20 421.3 12-20

C₂₁H₂₁N₃O₃ 364.14 364.2 12-21

C₂₂H₂₁N₃O₃ 376.14 376.4 12-22

C₂₃H₂₆N₄O₃ 407.18 407.1 12-23

C₂₀H₁₉N₃O₃ 350.13 350.1 12-24

C₂₃H₂₆N₄O₃ 407.18 407.2 12-25

C₂₀H₁₉N₃O₃ 350.13 350.2 12-26

C₂₅H₃₀N₄O₃ 435.22 435.1 12-27

C₂₂H₂₃N₃O₃ 378.16 378.1 12-28

C₂₅H₃₀N₄O₃ 435.22 435.2 12-29

C₂₂H₂₃N₃O₃ 378.16 378.2 12-30

C₂₂H₂₃N₃O₃ 378.16 378.2 12-31

C₂₂H₂₃N₃O₃ 378.16 378.2 12-32

C₂₂H₂₃N₃O₃ 378.16 378.1 12-33

C₂₂H₂₃N₃O₃ 378.16 378.2 12-34

C₂₀H₁₉N₃O₂S 366.10 366.1 12-35

C₁₉H₁₇N₃O₂S 352.09 352.3 12-36

C₂₅H₂₇FN₄O₃ 451.19 451.2 12-37

C₂₂H₂₀FN₃O₃ 394.13 394.2 12-38

C₂₃H₂₆N₄O₃ 407.18 407.3 12-39

C₂₀H₁₉N₃O₃ 350.13 350.1 12-40

C₂₂H₂₂N₄O₃ 391.15 391.1 12-41

C₂₃H₂₁N₅O₃ 416.14 416.1 12-42

C₂₁H₂₀N₄O₃S 409.10 409.1 12-43

C₂₁H₂₀N₄O₃S 409.10 409.3 12-44

C₂₁H₂₀N₄O₃S 409.10 409.3 12-45

C₂₁H₂₀N₄O₅S 441.09 441.1 12-46

C₂₀H₁₈N₄O₃ 363.11 363.1 12-47

C₂₁H₁₇N₅O₃ 388.11 388.0 12-48

C₂₀H₁₇FN₄O₃ 381.10 381.0 12-49

C₂₂H₂₄N₄O₄S 441.14 441.2 12-50

C₁₉H₁₇N₃O₄S 384.08 384.0 12-51

C₂₂H₂₄N₄O₂S 409.15 409.3 12-52

C₂₅H₂₈N₄O₃ 433.20 433.2 12-53

C₂₅H₂₈N₄O₃ 433.20 433.5 12-54

C₂₂H₂₁N₃O₃ 376.14 376.5 12-55

C₂₂H₂₂N₄O₃ 391.15 391.1 12-56

C₂₂H₂₂N₄O₃ 391.15 391.0 12-57

C₂₃H₂₄N₄O₃ 405.16 405.2 12-58

C₂₂H₂₁FN₄O₃ 409.14 409.3 12-59

C₂₂H₂₂N₄O₃ 391.15 391.1 12-60

C₂₂H₂₂N₄O₄ 407.14 407.1 12-61

C₂₂H₂₂N₄O₄ 407.14 407.1 12-62

C₂₃H₂₄N₄O₄ 421.16 421.1 12-63

C₂₁H₂₀N₄O₄ 393.12 393.1 12-64

C₂₁H₂₀N₄O₃S 409.10 409.1 12-65

C₂₄H₂₇N₅O₃S 466.16 466.1 12-66

C₂₁H₂₀N₄O₃S 409.10 409.1 12-67

C₂₃H₂₄FN₅O₃ 438.16 438.1 12-68

C₂₁H₂₀N₄O₃ 377.13 377.1 12-69

C₂₀H₁₈N₄O₃ 363.11 363.0

TABLE 13

Ex Calc Found No. R R¹ R² R³ X¹ X² Formula [M + H]⁺ [M + H]⁺ 13-1

Cl H N CH C₁₉H₁₇ClN₄O₂S 401.06 401.1 13-2

Cl H CH CH C₂₁H₁₉ClN₄O₄ 427.09 427.0 13-3

F H CH CH C₂₁H₁₉FN₄O₄ 411.12 411.0 13-4

H H CH CH C₂₃H₂₆N₄O₂S 423.16 423.1 13-5

F H CH CH C₂₃H₂₅FN₄O₂S 441.15 441.2 13-6

Cl H CH CH C₂₃H₂₅ClN₄O₂S 457.12 457.1 13-7

F H CH CH C₂₀H₁₈FN₃O₂S 384.09 384.1 13-8

Cl H CH CH C₂₀H₁₈ClN₃O₂S 400.07 400.1 13-9

H H N CH C₁₉H₁₈N₄O₂S 367.10 367.0 13-10

H H CH CH C₂₀H₂₁N₅O₃ 380.15 380.2 13-11

H H N CH C₂₁H₂₂N₄O₃ 379.15 379.0 13-12

H H N CH C₂₁H₂₂N₄O₃ 379.15 379.0 13-13

H H N CH C₂₁H₂₂N₄O₃ 379.15 379.0 13-14

H Cl CH CH C₂₁H₂₀ClN₃O₃ 398.10 398.3 (racemic) 13-15

H H CH CH C₂₁H₂₁N₃O₃ 364.14 364.5 (racemic) 13-16

H H N N C₂₀H₁₉N₅O₃ 378.13 378.5 13-17

Cl H N N C₂₀H₁₈ClN₅O₃ 412.09 412.4 13-18

F H N N C₂₀H₁₈FN₅O₃ 396.12 396.2 13-19

H H N CH C₂₀H₁₉N₅O₄ 394.12 394.1 13-20

Cl H CH N C₂₀H₁₈ClN₅O₄ 428.08 428.0 13-21

F H CH N C₂₀H₁₈FN₅O₄ 412.11 412.1 13-22

Cl H N CH C₂₀H₁₈ClN₅O₄ 428.08 428  

TABLE 14

Calc Found Ex No. R¹ R² R³ X¹ X² Formula [M + H]⁺ [M + H]⁺ 14-1

Cl H CH CH C₂₂H₂₂ClN₃O₃ 412.12 412.0 14-2

H H N CH C₂₁H₂₂N₄O₃ 379.15 379.1 14-3

Cl H CH CH C₂₅H₂₉ClN₄O₃ 469.18 469.1 14-4

F H CH CH C₂₅H₂₉FN₄O₃ 453.21 453.1 14-5

F H CH CH C₂₂H₂₂FN₃O₃ 396.15 396.1 14-6

H H CH N C₂₁H₂₂N₄O₃ 379.15 379.1 14-7

Cl H CH N C₂₁H₂₁ClN₄O₃ 413.11 413.0 14-8

F H CH N C₂₁H₂₁FN₄O₃ 397.14 397.1 14-9

F H N CH C₂₁H₂₁FN₄O₃ 397.14 397.1 14-10

Cl H CH N C₂₄H₂₈ClN₅O₃ 470.17 470.1 14-11

F H CH N C₂₄H₂₈FN₅O₃ 454.20 454.1 14-12

H H N N C₂₀H₂₁N₅O₃ 380.15 380.2

TABLE 15

Calc Found Ex No. R¹ R² R³ X¹ X² Formula [M + H]⁺ [M + H]⁺ 15-1

Cl H CH CH C₂₂H₂₀ClN₃O₃ 410.10 409.9 15-2

F H CH CH C₂₂H₂₀FN₃O₃ 394.13 393.4 15-3

H H N N C₂₀H₁₉N₅O₃ 378.13 378.1 15-4

Cl H CH CH C₂₅H₂₇ClN₄O₃ 467.16 467.1 15-5

F H CH CH C₂₅H₂₇FN₄O₃ 451.19 451.1 15-6

H H CH N C₂₄H₂₇N₅O₃ 434.20 434.1 15-7

H H CH N C₂₁H₂₀N₄O₃ 377.14 377.2 15-8

H H N CH C₂₁H₂₀N₄O₃ 377.14 377.0 15-9

Cl H N N C₂₀H₁₈ClN₅O₃ 412.09 412.2 15-10

F H N N C₂₀H₁₈FN₅O₃ 396.12 396.1

TABLE 16

Calc Found Ex No. R¹ R² R³ X¹ X² Formula [M + H]⁺ [M + H]⁺ 16-1

Cl H CH CH C₂₃H₂₅ClN₄O₃ 441.15 441.2 16-2

Cl H CH CH C₂₀H₁₈ClN₃O₃ 384.09 384.1 16-3

F H CH CH C₂₀H₁₈FN₃O₃ 368.12 368.2 16-4

H H N CH C₁₉H₁₈N₄O₃ 351.12 351.0 16-5

H H CH N C₁₉H₁₈N₄O₃ 351.12 351.0 16-6

H H CH N C₂₂H₂₅N₅O₃ 408.18 408.1 16-7

F H CH CH C₂₃H₂₅FN₄O₃ 425.18 425.2

TABLE 17

Calc Found Ex No. R R¹ R² R³ X¹ X² Formula [M + H]⁺ [M + H]⁺ 17-1

H H CH CH C₁₉H₁₈N₄O₃ 351.12 351.0 17-2

H H CH CH C₁₉H₁₈N₄O₃ 351.12 351.2

TABLE 18

Calc Found Ex No. R¹ R² R³ R⁴ R⁵ R⁶ X¹ X² Formula [M + H]⁺ [M + H]⁺ 18-1

Cl H H H Me CH CH C₂₄H₂₇ClN₄O₃ 455.16 455.0 18-2

H F H F H CH CH C₂₃H₂₄F₂N₄O₃ 443.17 443.2 18-3

F H H F H CH CH C₂₃H₂₄F₂N₄O₃ 443.17 443.0 18-4

H H H H Me CH CH C₂₄H₂₈N₄O₃ 421.2  421.0 18-5

H H H H Me CH CH C₂₁H₂₁N₃O₃ 364.14 364 18-6

Cl H H H Me CH CH C₂₁H₂₀ClN₃O₃ 398.10 398.0 18-7

F H H H Me CH CH C₂₁H₂₀FN₃O₃ 382.13 382.0 18-8

Me H H H H CH CH C₂₄H₂₈N₄O₃ 421.2  421.0 18-9

Me H H H H CH CH C₂₁H₂₁N₃O₃ 364.14 364.0 18-10

H H H H SMe CH CH C₂₁H₂₁N₃O₃S 396.11 396.0 18-11

H H H H NMe₂ CH CH C₂₂H₂₄N₄O₃ 393.17 393.0 18-12

H H H H CF₃ CH CH C₂₄H₂₅F₃N₄O₃ 475.17 475.0 18-13

H H H H CF₃ CH CH C₂₁H₁₈F₃N₃O₃ 418.11 418.0 18-14

H H H H Cl CH CH C₂₀H₁₈ClN₃O₃ 384.09 384.2

Biological Assays

The compounds of the disclosure have been characterized in one or moreof the following biological assays.

Assay 1: Biochemical JAK and Tyk2 Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

Serially or discretely diluted compounds were pre-incubated with each ofthe four JAK enzymes and the substrate in white 384-well microplates(Corning) at ambient temperature for 1 h. ATP was subsequently added toinitiate the kinase reactions in 10 μL total volume, with 1% DMSO. Thefinal enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM,1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrationsused are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrateconcentration is 200 nM for all four assays. Kinase reactions wereallowed to proceed for 1 hour at ambient temperature before a 10 μLpreparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1(pTyr701) antibody (Life Technologies, 2 nM final concentration) inTR-FRET dilution buffer (Life Technologies) was added. The plates wereallowed to incubate at ambient temperature for 1 h before being read onthe EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495nm) were recorded and utilized to calculate the percent inhibitionvalues based on DMSO and background controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC₅₀)and subsequently converted to pKi (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

Assay 2: Cellular JAK3 Potency Assay: Inhibition of IL-2 StimulatedpSTAT5 in Tall-1 T Cells

The potency of test compounds for inhibition of interleukin-2 (IL-2)stimulated STAT5 phosphorylation was measured in the Tall-1 human T cellline (DSMZ) using AlphaLisa. Because IL-2 signals through JAK3, thisassay provides a measure of JAK3 cellular potency.

Phosphorylated STAT5 was measured via the AlphaLISA SureFire UltrapSTAT5 (Tyr694/699) kit (PerkinElmer).

Human T cells from the Tall-1 cell line were cultured in a 37° C., 5%CO₂ humidified incubator in RPMI (Life Technologies) supplemented with15% Heat Inactivated Fetal Bovine Serum (FBS, Life Technologies), 2 mMGlutamax (Life Technologies), 25 mM HEPES (Life Technologies) and 1×Pen/Strep (Life Technologies). Compounds were serially diluted in DMSOand dispensed acoustically to empty wells. Assay media (phenol red-freeDMEM (Life Technologies) supplemented with 10% FBS (ATCC)) was dispensed(4 μL/well) and plates shaken at 900 rpm for 10 mins. Cells were seededat 45,000 cells/well in assay media (4 μL/well), and incubated at 37°C., 5% CO₂ for 1 hour, followed by the addition of IL-2 (R&D Systems;final concentration 300 ng/ml) in pre-warmed assay media (4 μL) for 30minutes. After cytokine stimulation, cells were lysed with 6 ul of 3×AlphaLisa Lysis Buffer (PerkinElmer) containing 1× PhosStop and Completetablets (Roche). The lysate was shaken at 900 rpm for 10 minutes at roomtemperature (RT). Phosphorylated STAT5 was measured via the pSTAT5AlphaLisa kit (PerkinElmer). Freshly prepared acceptor bead mixture wasdispensed onto lysate (54) under green filtered <100 lux light. Plateswere shaken at 900 rpm for 2 mins, briefly spun down, and incubated for2 hrs at RT in the dark. Donor beads were dispensed (54) under greenfiltered <100 lux light. Plates were shaken at 900 rpm for 2 minutes,briefly spun down, and incubated overnight at RT in the dark.Luminescence was measured with excitation at 689 nm and emission at 570nm using an EnVision plate reader (PerkinElmer) under green filtered<100 lux light.

To determine the inhibitory potency of test compounds in response toIL-2, the average emission intensity of beads bound to pSTAT5 wasmeasured in a human T cell line. IC₅₀ values were determined fromanalysis of the inhibition curves of signal intensity versus compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC₅₀) values (mean±standard deviation).

Assay 3: Inhibition of IL-2 Stimulated pSTAT5 in CD4+ T Cells Isolatedfrom Murine Splenocytes

The potency of test compounds for inhibition of interleukin-2 (IL-2)stimulated STAT5 phosphorylation was measured in the CD4+ T cellsisolated from murine splenocytes using AlphaLisa. Because IL-2 signalsthrough JAK3, this assay provides a measure of JAK3 cellular potency inmouse.

Phosphorylated STAT5 was measured via the AlphaLISA SureFire UltrapSTAT5 (Tyr694/699) kit (PerkinElmer).

CD4+ T cells were isolated from murine splenocytes via negativeselection on a magnetic column (Miltnyi Biotec) and re-suspended inassay media (phenol red-free DMEM (Life Technologies) supplemented with10% FBS (ATCC)). Cells were seeded at 50,000 cells/well in assay media(2 μL/well). Compounds were serially diluted in DMSO and diluted to 2×final concentration in assay media. Compound was added (4 μL/well) andthe cells incubated at 37° C., 5% CO₂ for 1 hour, followed by theaddition of IL-2 (R&D Systems; final concentration 7 ng/ml) inpre-warmed assay media (2 μL) for 30 minutes. After cytokinestimulation, cells were lysed with 2 μl of 5× AlphaLisa Lysis Buffer(PerkinElmer). The lysate was shaken at 900 rpm for 10 minutes at roomtemperature (RT). Phosphorylated STAT5 was measured via the pSTAT5AlphaLisa kit (PerkinElmer). Freshly prepared acceptor bead mixture wasdispensed onto lysate (5 ul) under green filtered <100 lux light. Plateswere shaken at 900 rpm for 2 mins, briefly spun down, and incubated for2 hrs at RT in the dark. Donor beads were dispensed (5 μl) under greenfiltered <100 lux light. Plates were shaken at 900 rpm for 2 mins,briefly spun down, and incubated overnight at RT in the dark.Luminescence was measured with excitation at 689 nm and emission at 570nm using an EnVision plate reader (PerkinElmer) under green filtered<100 lux light.

To determine the inhibitory potency of test compounds in response toIL-2, the average emission intensity of beads bound to pSTAT5 wasmeasured in primary CD4+ T cells isolated from murine splenocytes. IC₅₀values were determined from analysis of the inhibition curves of signalintensity vs compound concentration. Data are expressed as pIC₅₀(negative decadic logarithm IC₅₀) values (mean±standard deviation).

Compounds 1-10, 19, 20, 21, and 22 all had pIC₅₀ values over 6.0 in thisassay.

Assay 4: Inhibition of IL-2 Stimulated pSTAT5 in Human PBMC CD4+ T Cells

The potency of test compounds for inhibition of interleukin-2 (IL-2)stimulated STAT5 phosphorylation was measured in human peripheral bloodmononuclear cell (PBMC) CD4+ T cells using flow cytometry. Because IL-2signals through JAK3, this assay provides a measure of JAK3 cellularpotency in human primary cells.

Phosphorylated STAT5 was measured via flow cytometry by measuring pSTAT5response in CD4+ T cells (Becton Disckinson, AlexaFluor 647 Mouseanti-Human STAT5 (pY694) and PE Mouse anti-Human CD4).

Human PBMCs were isolated from donors via Ficoll gradient and frozen at−80° C. Frozen PBMCs were thawed and cultured for one hour in a 37° C.,5% CO₂ humidified incubator in RPMI (Life Technologies) supplementedwith 10% Heat Inactivated Fetal

Bovine Serum (FBS, Life Technologies), 2 mM Glutamax (LifeTechnologies), 25 mM HEPES (Life Technologies) and 1× Pen/Strep (LifeTechnologies). Cells were seeded into 2 ml deep well plates at 4e6cells/ml (50 μl/well). Compounds were serially diluted in DMSO anddiluted to 2× final concentration in assay media. Compound was added(100 μl/well) and the cells incubated at 37° C., 5% CO₂ for 1 hr,followed by the addition of IL-2 (R&D Systems; final assay concentration100 ng/ml) in pre-warmed assay media (50 μL/well) for 30 minutes. Aftercytokine stimulation, cells were fixed for 10 mins at 37° C. (BectonDickinson). Cells were washed and re-suspended in Dulbecco's phosphatebuffered saline (DPBS, Life Technologies). Cells were permeabilized for30 mins with ice cold Perm Buffer III (Becton Dickinson), and thenwashed and re-suspended with DPBS containing 2% fetal bovine serum(staining buffer, Life Technologies). Cells were stained for 1 hr with a50× dilution of CD4 surface marker (PE Mouse anti-Human CD4) and 5×dilution of pSTAT5 antibody (Becton Disckinson, AlexaFluor 647 Mouseanti-Human STAT5 (pY694)) in staining buffer. Cells were washed andre-suspended in staining buffer before storage at 4° C.

To determine the inhibitory potency of test compounds in response toIL-2, the median fluorescence intensity of pSTAT5 in CD4+ gated cellswas measured in human PBMCs with a BD LSRII with analysis in FCS Express6. IC₅₀ values were determined from analysis of the inhibition curves ofsignal intensity vs compound concentration. Data are expressed as pIC₅₀(negative decadic logarithm IC₅₀) values (mean±standard deviation).

Compounds 1-6, 8 and 9 were tested in this assay and exhibited valuesvery similar to the ones obtained in assay 2 (IL-2 Stimulated pSTAT5 inTall-1 T cells assay).

Assay 5: JAK Cytotoxicity Assay

A CellTiter-Glo luminescent cell viability/cytotoxicity assay wascarried out in BEAS-2B human lung epithelial cells (ATCC) under thenormal growth condition.

Cells were grown at 37° C. in a 5% CO₂ humidified incubator in 50%DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 500 cells/well density in white 384-welltissue culture plates (Corning) with 25 μL medium, and were allowed toadhere overnight in the incubator. On day 2 of the assay, 5 μL of mediumcontaining dose-responses of test compounds was added, and incubated at37° C. for 48 h. 30 μL of CellTiter-Glo detection solution (Promega) wassubsequently added, mixed on an orbital shaker for 5 min, and incubatedfor additional 10 min before being read on the EnVision reader.Luminescence signals were recorded and percent DMSO control values werecalculated.

For dose-response analysis, percent DMSO control data were plotted vs.compound concentrations to derive dose-response curves by lineconnecting each data point. The concentration at which each curvecrosses the 15% inhibition threshold is defined as CC₁₅. Results wereexpressed as the negative logarithm of the CC₁₅ value, pCC₁₅.

It is expected that test compounds exhibiting a lower pCC₁₅ value inthis assay have less likelihood to cause cytotoxicity. Compounds of thedisclosure tested in this assay typically exhibited pCC₁₅ values between5 and about 6.

Assay 6: Caco-2 Permeation Assay

The Caco-2 permeation assay was performed to model the ability of testcompounds to pass through the intestine and get into the blood streamafter oral administration. The rate at which test compounds in solutionpermeate a cell monolayer designed to mimic the tight junction of humansmall intestinal monolayers was determined.

CacoReady 24-well transwell plates were obtained from ADMEcell (Alameda,Calif.). The compounds were evaluated at a concentration of 5 μM from 10mM DMSO stock solutions in duplicate (n=2). The passive permeability ofthe compounds tested was evaluated using Caco-2 cell monolayers alongwith Verapamil (25 μM) to inhibit P-gp transport proteins in the apicalto basolateral (A-B) direction. The experiment was conducted in a 37°C., 5% CO₂ incubator. Caco-2 culture media consisted of standardfiltered DMEM, FCS 10%, L-Glutamine 1% and PenStrep 1%. Basal assayplate was prepared by adding 750 μL, of transport buffer to A-B wells. ACacoReady™ plate was prepared by removing the Caco-2 media from theapical wells and replacing with fresh transport media (200 μL repeatedfor a total of 3 washes). Blank media (200 μL) was then replaced withdiluted compound for A-B wells. To begin the incubation, the basal platewas removed from the incubator and the apical section was added on topof it. Samples (40 μL) were collected from the apical and basalcompartments for time zero (t0). Samples were collected again after 120minutes (t120) from the apical and basal compartments. All samples werediluted and prepared for bioanalysis by LC-MS/MS. The permeationcoefficient (K_(p), mean A to B+Verapamil Papparent) in cm/sec wascalculated as dQ (flux)/(dt×Area×concentration).

In this assay, a K_(p) value less than about 5×10⁻⁶ cm/sec is consideredfavorable to minimize systemic exposure and target the colon. A K_(r)value less than about 10×10⁻⁶ cm/sec may also be sufficient to minimizesystemic exposure and target the colon. By comparison, PF-06651600, aJAK3 inhibitor available systemically (2-propen-1-one,1-[(2S,5R)-2-methyl-5-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-1-piperidinyl])exhibited a Kp value of 25.

In Vitro Assay Results

All of the compounds of Examples 1 to 18 and Tables 1 to 12 were testedin one or more of the assays described above.

In Table 13 below, for the JAK1, JAK 2, JAK3, and TYK2 enzyme assays, Arepresents a pK_(i) value≥10 (K_(i)≤0.1 nM), B represents a pK_(i) valuebetween 9 and 10 (K_(i) between 1 nM and 0.1 nM), C represents a pK_(i)value between 8 and 9 (K_(i) between 10 nM and 1 nM), D represents apK_(i) value between 7 and 8 (K_(i) between 100 nM and 10 nM), and Erepresents a pK_(i) value of 7 or below (K₁ of 100 nM or above). For theTall-1 Potency assay, A represents a pIC₅₀ value≥7.5 (IC₅₀≤32 nM), Brepresents a pIC₅₀ value between 6.7 (included) and 7.5 (IC₅₀ between200 nM and 32 nM), and C represents a pIC₅₀ value between 6 and 6.7(IC₅₀ between 1 μM and 200 nM). For the JAK3 (pKi)-JAK1 (pKi) values, Arepresents a value of 3 or above, B represents a value of 2.3 to 3 and Crepresents a value of 1.8 to 2.3. For the Caco assay, A represents avalue below 5×10⁻⁶ cm/sec, B represents a value between 5×10⁻⁶ and10×10⁻⁶ cm/sec, C represents a value between 10×10⁻⁶ and 22×10⁻⁶ cm/sec.

TABLE 13 JAK3 Caco JAK JAK JAK Tyk (pKi)- K_(p) Example 1 2 3 2 Tall-1JAK1 10⁻⁶ Number (pKi) (pKi) (pKi) (pKi) (pIC₅₀) (pKi) cm/sec  1 E D A EB A A  2 E E A E A A A  3 E E B E B A A  4 E E A E B A A  5 E D A E B AA  6 E D A E A A A  7 E D B E B A B  8 E D A E A A A  9 E B B A A 10 E EA E B A A 11 E E B E B A A 12 E E B E B B A 13 E E A E B A A 14 E E B EB A C 15 E B C A A 16 E B B A A 17 E B C B 18 E D B E A A C 19 E E A E BA A 20 E E A E A A A 21 E E A E B A B 22 E E A E B A A 23 E D A E B A A24 E E B E A A A 1-1  E D B E A 1-2  E D A E A A C 1-3  E B B A 1-4  E BC A 1-5  E D A E A A B 1-6  E B C A 1-7  E B C B 1-8  E E B E B A B 1-9 E A C A 1-10 E B C A 1-11 E E A E A A C 1-12 E B B A 1-13 E B B B A 1-14E A B A A 1-15 E E A E B A A 1-16 E D B E B B A 1-17 E D B E A A A 1-18D D B E A B A 1-19 E D B E B A A 1-20 E D B E A B A 1-21 E E B E B A A1-22 E D A E B A A 1-23 D B A B A 1-24 E E B E C A C 1-25 E B B A C 1-26E E A E B A B 1-27 E D A E B A 1-28 E B C B 1-29 D D B E B B 1-30 D A BB 1-31 E E A E B A 1-32 E B C A 1-33 E E B E B A 1-34 E D B E A A 1-35 DA A A 1-36 D D B E A B 1-37 E B C A 1-38 E E A E B A 1-39 E B B A B 1-40E E B E B B 1-41 E E B E B A 1-42 E B C A B 1-43 E A A A 1-44 E E A E AA C 1-45 E E A E A A 1-46 E E B E C A 1-47 E E B E B B C 1-48 E C B B2-1  D A C B 2-2  E D B E B A A 2-3  E D B E B A B 2-4  E B C B 2-5  E DB E B A B 2-6  E A C A C 2-7  E B B A A 2-8  E D B E B A A 2-9  E B B BA 2-10 E D B E A A A 2-11 E E B E B A B 2-12 E A C A C 2-13 E B C B 2-14E A A A 2-15 E E A E B A C 2-16 E B C A 2-17 E E A E B A 3-1  E E B E AA A 3-2  E E B E B A A 3-3  E E B E A A B 3-4  E D B E A A B 3-5  D D BE A B A 3-6  D A A A 3-7  E D A E B A A 3-8  E E B E A A A 3-9  E B B BA 3-10 E B A A A 3-11 E D A E B A A 3-12 E B B B A 3-13 E D B E A A A3-14 E B B A 3-15 E E A E B A A 3-16 E A B A A 3-17 E D A E A A A 3-18 EA B A A 3-19 E E B E B A A 3-20 E D B E B B A 3-21 E E B E A A A 3-22 EE A E B A A 3-23 E D A E B A A 3-24 E E B E B A A 3-25 E C B B A 3-26 EB B A A 3-27 E E A E B A A 3-28 E A A A B 3-29 E B A A A 3-30 E D A E BA A 3-31 E E A E B A B 3-32 E E B E A A A 3-33 E E B E B B A 3-34 E E AE B A A 3-35 E E B E A A A 3-36 E E A E B A A 3-37 E D A E A A A 3-38 EE A E B A A 3-39 E E A E B A A 3-40 B D A E A A A 3-41 B C B E A B A3-42 B D B E A B A 3-43 E D B E A A A 3-44 D D B E A B A 3-45 E E B E AA A 3-46 E E A E A A A 3-47 E D B E A B A 3-48 D D A E A A A 3-49 E D BE A B A 3-50 E E B E B A A 3-51 E E B E C A A 3-52 E E B E B A A 3-53 EE B E B A A 3-54 E E B E A A A 3-55 E E A E B A A 3-56 E D A E A A A3-57 E E A E B A A 3-58 E E A E B A A 3-59 E D B E A B A 3-60 E E A E BA 3-61 E D A E A A A 3-62 E D B E A A A 3-63 E E A E B A A 3-64 E D A EA A A 3-65 E D B E A A A 3-66 E E B E A A A 3-67 E E B E B A A 3-68 D EB E C B A 3-69 E E A E B A A 3-70 E E A E B A A 3-71 E E A E A A A 3-72E E B E A A A 3-73 E E A E A A A 3-74 E E A E A A A 3-75 E E B E A A A3-76 E A B A A 3-77 E E A E B A A 3-78 E E B E B A A 3-79 E E B E B A3-80 E E A E B A A 3-81 E E A E A A A 3-82 D A A B A 3-83 E E A E A A B3-84 E A B A 3-85 E D A E A A A 3-86 E E A E B A 3-87 E E B E B A 3-88 EE B E B A 3-89 E E A E B A 3-90 D A A B 3-91 D D A E A A 3-92 E E A E AA 3-93 E E A E A A 3-94 E E A E A A 3-95 E E A E A A 3-96 E E B E B A3-97 E E B E A B 3-98 E E A E B A 3-99 E E A E A A  3-100 E E B E A A 3-101 E D A E A A  3-102 E E B E B A  3-103 E D A E A A  3-104 E E A EA A  3-105 E D A E A A  3-106 E E B E B A  3-107 E E A E A A  3-108 E DA E A A  3-109 E E B E B A  3-110 E E B E B A  3-111 E E B E C A  3-112E E B E B A A  3-113 E D A E A A A  3-114 E E B E A A  3-115 E B B E B AA  3-116 E D B E B B  3-117 E D B E B B  3-118 E E B E A A  3-119 E D AE B B A  3-120 E D A E B A A  3-121 E D B E B A A  3-122 E D A E A A 3-123 E A C A  3-124 E B B A  3-125 E E B E B A  3-126 E D A E C A 3-127 E D A E B A  3-128 E B A E B A  3-129 E B C A A  3-130 E B B A A 3-131 E B B E A B  3-132 E E B E C A  3-133 E D A E C A  3-134 E B B A4-1  E E B E B A A 4-2  D D B E B B A 4-3  E B B B A 4-4  E E A E B A A4-5  E E B E A A A 4-6  E E B E B A A 4-7  E E C E B B A 4-8  E B B B5-1  E B C A 5-2  E E B E B A A 5-3  E E A E A A A 5-4  E E B E A A A5-5  E D A E B A A 5-6  E E B E B A A 5-7  E D A E B A A 5-8  E B C A5-9  E B C A A 5-10 E B B A A 5-11 E B B A A 5-12 E E B E B A A 5-13 E BB B A 5-14 E E A E A A A 5-15 E A B A A 5-16 E 6 A E A A A 5-17 E E B EA A A 5-18 E E A E A A A 5-19 E E B E B A A 5-20 E B C A 5-21 E E B E AA 5-22 E E B E B A 5-23 E E B E B A 5-24 E B B A 5-25 E B A A 5-26 E B CB 6-1  D A B B 6-2  D D A E A B C 6-3  E D B E A A C 6-4  E D A E B A A6-5  E B A A A 6-6  E D B E A A A 6-7  E B A A B 6-8  D A A B 6-9  E B BA A 6-10 E D B E A A C 6-11 E A B A B 6-12 E B B A A 6-13 E D B E A B A6-14 D A A B B 6-15 E D B E B A B 6-16 E D B E B A A 6-17 E E A E B A A6-18 E E A E B A B 6-19 D B B B 6-20 D C B E B B A 6-21 D D B E B B 6-22E B C A 6-23 E B C A 7-1  E B C A 7-2  E D B E A A A 7-3  E E B E B A C7-4  D C B E A B C 7-5  D B B C 7-6  D A A B 7-7  E D B E B A A 7-8  E EA E B A A 7-9  E D A E A A A 7-10 E D B E B A C 7-11 E D A E A A 7-12 ED B E B A 7-13 E D A E A A 7-14 E D B E A B 8-1  E D B E A B C 8-2  E DB E B A A 8-3  E D B E A A A 8-4  E D B E B B A 8-5  E D A E A A A 8-6 E B B A B 8-7  E D B E B A B 8-8  E A B A B 8-9  E B C B 8-10 D A A A8-11 D D A E A B 8-12 E B C A 8-13 E B C A 8-14 E E A E B A C 9-1  E D AE A A C 9-2  E E B E A A B 9-3  E B C C 9-4  E C A E A A C 9-5  D B C C9-6  E B A A C 9-7  E A B A 9-8  D B C C 9-9  D C A D A B 9-10 E B A A C9-11 E A B A C 10-1  D A B B A 10-2  D C A D A B C 10-3  D B B B 10-4  ED B E B A A 10-5  D B B C 10-6  D B A B 10-7  E D B E B A B 11-1  E B AA A 11-2  E B C A 12-1  E D B E B A 12-2  E B B B 12-3  D B C B 12-4  EE B E B A B 12-5  E B B B 12-6  D B B B 12-7  D B C C 12-8  E B C A A12-9  E B C A A 12-10  E C B B 12-11  E B C A 12-12  E B B A 12-13  E BC A 12-14  E B C C 12-15  E B B B 12-16  D C B E A B 12-17  D C A E A B12-18  D A B B 12-19  E A C A 12-20  E D B E A A B 12-21  E B A B A12-22  E B C A A 12-23  E B B A B 12-24  E B C A A 12-25  E B C B A12-26  E D B E C B 12-27  E D A E A A C 12-28  E C A E B A C 12-29  E DB E A A C 12-30  E C A E A A C 12-31  E C A E B A C 12-32  E D A E A A C12-33  E E B E B A A 12-34  E E B E A B A 12-35  E E B E B B A 12-36  DE A E A A A 12-37  D B B B A 12-38  E E A E B A A 12-39  E E B E A A B12-40  E B C B A 12-41  E E B E A A A 12-42  E D B E B B A 12-43  E D AE A A A 12-44  E D A E B A A 12-45  E E A E C A A 12-46  E D A E B A A12-47  E E B E B A A 12-48  E E B E A A A 12-49  E E B E C A 12-50  E EA E B A 12-51  D D A E B A 12-52  D B C B 12-53  E C B E A B 12-54  E DB E A B 12-55  E A B A 12-56  E A B A 12-57  E D B E B A 12-58  E D A EB A 12-59  E A B A 12-60  E E B E B A 12-61  E E A E B A 12-62  E A B A12-63  E E B E B A 12-64  E E A E A A 12-65  E E B E C A 12-66  E E B EB A 12-67  E E B E B A 12-68  E E B E C A 12-69  E A B A 13-1  E E B E BA A 13-2  E E B E A A A 13-3  E A A A A 13-4  E D A E B A 13-5  E D A EB A 13-6  E E A E A A 13-7  E D A E B A 13-8  E E B E A A 13-9  E E B EB A 13-10  E E B E B A 13-11  E E A E A A C 13-12  E E B E B A C 13-13 E E B E C A C 13-14  E D A E A A 13-15  E D B E B A 13-16  E E B E B A C13-17  E A B A 13-18  E B B A 13-19  E E A E B A 13-20  E E B E B B13-21  E D B E A B 13-22  E E B E B A 14-1  E E B E A A A 14-2  E E A EB A A 14-3  E B B B 14-4  D B B B 14-5  D B A B 14-6  D A A B 14-7  D AA B 14-8  D A A B 14-9  E E B E B A 14-10  D B C C 14-11  D B C B 14-12 E E B E C A 15-1  E E B E A A A 15-2  D D A E A B A 15-3  E E A E A A A15-4  E A B A 15-5  D D B E B B 15-6  D A C B 15-7  E D B E A B 15-8  DB B B 15-9  E E A E B A 15-10  E E A E A A 16-1  E E A E B A A 16-2  E EA E A A A 16-3  E E B E A A A 16-4  E B B A A 16-5  E E A E A A A 16-6 E E B E C A 16-7  E E A E B A 17-1  E E B E B A A 17-2  E A C A A 18-1 E D B E A B A 18-2  D A A C 18-3  D D A E A B 18-4  D A B B 18-5  D B AB 18-6  D B A B 18-7  D B A C 18-8  E E B E B A 18-9  E E A E A A 18-10 E D A E B A 18-11  E B C A 18-12  D D B E B B 18-13  E D A E A A 18-14 E B B A

Assay 7: Colon and Plasma Mouse Pharmacokinetics

6 male Balb/c mice were administered 10 mg/kg of compound in 1%HPMC+0.1% Tween-80 by PO administration. At 0.5, 2 and 6 hours afterdose administration, animals were anesthetized, and terminal bloodsamples were collected by cardiac puncture, followed by collection ofcolon contents and colon tissue.

Blood samples were collected into K₂EDTA and stored on wet ice untilprocessed to plasma by centrifugation (12000 rpm at 4° C.). Plasmasamples were transferred to cluster tubes and placed on dry ice prior tofreezer storage. The colon contents from each animal were collected ateach terminal blood collection time point. The colon tissues wereflushed with saline and patted dry. The colon and colon content tissueswere homogenized using sterile water containing 0.1% formic acid 9:1(water:tissue, v/w). The homogenized tissues and colon contents weretransferred to cluster tubes and placed on dry ice prior to freezerstorage. All samples were analyzed using LC/MS/MS against analyticalstandards.

The composite pharmacokinetic parameters of the compounds weredetermined by non-compartmental analysis using Phoenix WinNonlin Version6 (Certara, St. Louis, Mo.) and using mean values from 2 animals/timepoint. For plasma concentrations below the quantification limit (BQL),the lowest concentration measurable or the BLOQ (below limit ofquantification) was used.

A colon to plasma ratio was determined as the ratio of the colon AUC tothe plasma AUC. Compounds 1, 2, 3, 4, 6, 7, 8, 21 and 22 exhibited acolon to plasma ratio in excess of about 1250. Compounds 9, 5, 19, and20 exhibited a colon to plasma ratio in excess of about 200.

In contrast, the reference compound (PF-06651600, a JAK3 inhibitoravailable systemically) 2-propen-1-one,1-[(2S,5R)-2-methyl-5-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-1-piperidinyl],exhibited a colon to plasma ratio of 2.8

Assay 8: Mouse Model of Oxazolone-Induced Colitis

Oxazolone-induced colitis is an experimental model that has ahistological resemblance to human ulcerative colitis (Heller et al.Immunology, 2002, 17, 629-638). Adult BALB/C mice from Harlan were usedin the assay. On day 1, animals were lightly anesthetized withisoflurane and the hairs between the shoulder were carefully removedbefore oxazolone (4%, 150 μL, 4:1 acetone: olive oil formulation) orvehicle solution was slowly applied for skin sensitization. Seven daysafter skin sensitization, the mice were fasted around 6 hours prior,anesthetized with ketamine/xylazine injection, and a 1 mL syringeequipped with a 3.5-F catheter, filled with oxazolone solution, wasinserted carefully about 4 cm into the colon of the mouse. Followinginsertion, 50 μL of the oxazolone solution (0.8% in 1:1 ethanol:waterformulation) was injected very slowly (over 30 sec using an injectionpump) into the colon. Drug treatment (PO, QD or BID or TID) or vehiclewas initiated a day prior to the oxazolone intrarectal (IR) challenge.Two-day post oxazolone intrarectal challenge, the disease was assessedby treatment-blinded experimenters for each mouse according to thecriteria score: stool consistency score (0, normal; 2, loose; 4,diarrhea), gross bleeding score (0, absence; 2, blood tinged; 4,presence); Combined stool score endpoint=stool consistency score+stoolblood score.

Select compounds were tested in the assay. Efficacy in the model isevidenced by a statistically significant decrease in combined stoolscore endpoint as compared with the score from vehicle treated animals.

The compounds 1, 2, 3, 4, 5, 6, 7, 8, 3-11, 5-10, 19, 15-1, 3-55, 3-34,15-3, 21, 3-80, 3-81, 3-72 and 3-57 exhibited a statisticallysignificant decrease in combined stool score endpoint as compared withvehicle treated animals in the oxazolone model at a dose of 3 mg/kg (PO,BID). The compounds 3-113 and 3-74 exhibited a statistically significantdecrease in the combined stool score endpoint as compared with vehicletreated animals in the oxazolone model at a dose of 1 and 10 mg/kg only(PO, BID).

Assay 9: Immunosuppression Effects in Mouse Splenic Natural Killer (NK)Cells

Depletion of mouse splenic cells is an experimental model ofimmunosuppression (Kudlacz et al., Am. J. of Transplantation, 2004, 4,51-57). Select compounds were assessed in the mouse splenic cell modelfollowing the same treatment paradigm used in the oxazolone-inducedcolitis model (assay 8).

Adult male Balb/C mice from Harlan were used for the study. The testcompound and a Jak3 covalent inhibitor standard 2-proper-1-one,1-[(2S,5R)-2-methyl-5-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-1-piperidinyl](30mg/kg, BID) as a positive control, were dosed orally for three days tonaïve mice. Spleens were harvested up to 4 hours post last dose andcrushed immediately for cell subtype staining. Prior to fixation,fluorophore-labelled antibodies for CD19 (FITC; B cells), CD3e (PE; panT cells) and CD49e (APC; NK cells) were incubated with splenocytesamples from each animal to allow for simultaneous, multiple subtype %analysis on the flow cytometer. The number of total spleen cells foreach animal was measured by Scepter™ 2.0 Handheld Automated CellCounter.

The absolute number of lymphocyte subtype population (e.g., splenic B, Tand NK cells) was calculated from the percentage of each subtype timesthe total spleen cells for each animal. A one-way ANOVA, with Fisher'sLSD post hoc test, was used to compare the splenic lymphocytes number ofthe vehicle and test compound groups. The a level was set at p<0.05.Data were presented as the mean±SEM for each group.

The positive control PF-06651600, a JAK3 inhibitor availablesystemically (2-propen-1-one,1-[(2S,5R)-2-methyl-5-(7H-pyrrolo[2,3-d]pyridin-4-ylamino)-1-piperidinyl])(30 mg/kg PO, BID), significantly decreased splenic NK cell counts.Splenic NK cell counts were unaffected by compound 1 at (PO, BID) dosesup to 300 mg/kg (the maximum dose tested). Splenic NK cell counts wereunaffected by compounds 4, 6 and 8 at (PO, BID) doses up to 100 mg/kg(the maximum dose tested). Splenic NK cell counts were unaffected bycompound 3 at (PO, BID) doses up to 85 mg/kg (the maximum dose tested).Splenic NK cell counts were unaffected by compound 2 at (PO, BID) dosesup to 30 mg/kg (the maximum dose tested). Splenic NK cell counts wereunaffected by compound 5 at (PO, BID) doses up to 80 mg/kg (the maximumdose tested). No treatment effects were observed for the B and T cellpopulations with any of the compounds tested.

Assay 10: Systemic Target Engagement Assay: Murine Model of IL-2 InducedpSTAT5 Induction in Thymus

IL-2 is an important cytokine underlying the pathophysiology ofgastro-intestinal diseases such as irritable bowel disease (IBD; Guanand Zhang. Mediators Inflamm, 2017; 4810258). IL-2 binds to cell surfacereceptors activating members of the Janus family of kinases (JAK),specifically JAK3, which then phosphorylates STAT5 and subsequentlyactivates further transcription pathways. In this model, a dose of IL-2was delivered systemically to mice to induce the phosphorylation ofSTAT5 (pSTAT5) which was then measured as the endpoint. The compoundstested in this assay which do not exhibit significant inhibition of IL-2induced pSTAT5 compared to the vehicle-treated;

IL-2 challenged control animals, demonstrate lack of systemic activity.Adult male Balb/c mice from Harlan were used in the assay. Animals wereadministered test compound via oral gavage (PO, 10 or 30 mg/kg forsingle dose studies; 10-60 mg/kg for dose response studies) on theafternoon of the day prior to study and then in the morning on the dayof study. Two hours after the second PO dose administration, mice wereinjected intraperitoneally (i.p.) with 100 μl of an appropriate IL-2dose (for a total of 1-5 μg/mouse depending upon IL-2 batch; R&DSystems). 90-120 minutes after IL-2 insult, thymus samples wereharvested. Phosphorylated STAT5 (pSTAT5) levels were determined in thethymus using AlphaLISA (AlphaLISA® SureFire® Ultra p-STAT5 (Tyr694/699)HV (high volume). Activity in the model is evidenced by a lack ofsignificant inhibition in the level of pSTAT5 present in the thymus oftreated animals at 1.5-2 hours following IL-2 challenge.

Compounds 6 and 7 were tested at 10 mg/kg and exhibited no significantinhibition of IL-2 induced pSTAT5 compared to the vehicle-treated; IL-2challenged control animals, therefore demonstrating lack of systemicactivity. Compound 1 was tested at 10, 30, 60 and 100 mg/kg andexhibited no significant inhibition of IL-2 induced pSTAT5 compared tothe vehicle. Compound 2 was tested at 10, 30 and 60 mg/kg and exhibitedno significant inhibition of IL-2 induced pSTAT5 compared to thevehicle. Compound 3 was tested at 30 and 100 mg/kg and exhibited nosignificant inhibition of IL-2 induced pSTAT5 compared to the vehicle.Compounds 8, 5 and 4 were tested at 30 mg/kg and exhibited nosignificant inhibition of IL-2 induced pSTAT5 compared to the vehicle.

In contrast, the reference compound (PF-06651600, a JAK3 inhibitoravailable systemically) 2-propen-1-one,1-[(2S,5R)-2-methyl-5-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-1-piperidinyl],exhibited significant inhibition of pSTAT5 induction in the thymus at10, 30 and 50 mg/kg, demonstrating systemic activity.

Crystal Structures

Co-crystal structures were obtained for compounds 1, 3 and 4 each boundto human JAK3. The resolutions for the complex structures were at 2.73Å, 2.80 Å, and 2.64 Å respectively for compound 1, compound 3 andcompound 4. In each case, the ligands were observed to bind in the ATPbinding site. Based on a distance of less than 3.5 Å of the donor andacceptor atoms four specific hydrogen bonds were identified for each ofthe ligands, namely to the main chain atoms of Glu903, Leu905 and Phe968as well as the side chain atoms of Glu871. An additional hydrogen bondwas identified for compound 1 to the side chain atoms of Asp 912. Ofparticular note, the ligands are each covalently bound to Cys909 of JAK3located shortly after the hinge region. The S—H moiety of the cysteineresidue performs an addition reaction to the Michael system. Theobserved results of the covalent binding interaction in the crystalstructures confirms the irreversible binding nature for each of theseligands to JAK3.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

1-46. (canceled)
 47. A method for treating celiac disease in a patient,the method comprising administering to the patient a compound of theformula:

or a pharmaceutically acceptable salt thereof.
 48. The method of claim47, wherein the compound or the pharmaceutically acceptable salt isadministered orally.
 49. The method of claim 47, wherein the patient isa human.
 50. The method of claim 47, where the compound is administeredas a crystalline freebase form.
 51. The method of claim 50, whereincrystalline freebase form is characterized by a powder X-ray diffractionpattern comprising diffraction peaks at 2θ values of 9.67±0.20,11.61±0.20, 17.61±0.20, 18.88±0.20, and 23.33±0.20.
 52. The method ofclaim 51, wherein the powder X-ray diffraction pattern further comprisesadditional diffraction peaks at 2θ values of 4.82±0.20, 15.69±0.20, and16.19±0.20.
 53. The method of claim 52, wherein the powder X-raydiffraction pattern further comprises two or more additional diffractionpeaks at 2θ values selected from 11.92±0.20, 12.98±0.20, 13.23±0.20,16.45±0.20, 16.67±0.20, 19.39±0.20, 19.96±0.20, 20.14±0.20, 22.14±0.20,23.84±0.20, 24.06±0.20, 24.29±0.20, 25.31±0.20, 25.63±0.20, 27.06±0.20,27.31±0.20, 30.10±0.20, and 30.53±0.20.
 54. The method of claim 50,wherein the crystalline freebase form is characterized by a powder X-raydiffraction pattern in which the peak positions are substantially inaccordance with the peak positions of the pattern shown in FIG.
 9. 55.The method of claim 50, wherein the crystalline freebase form ischaracterized by a differential scanning calorimetry trace recorded at aheating rate of 10° C. per minute which shows a maximum in endothermicheat flow at a temperature between 198° C. and 204° C.
 56. The method ofclaim 50, wherein the crystalline freebase form is characterized by adifferential scanning calorimetry trace recorded at a heating rate of10° C. per minute which shows a maximum in endothermic heat flow with apeak at 201.3° C.±2° C.
 57. The method of claim 50, wherein thecrystalline freebase form is characterized by a differential scanningcalorimetry trace substantially in accordance with that shown in FIG.10.
 58. A method for treating celiac disease in a patient, the methodcomprising administering to the patient a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound of theformula:

or a pharmaceutically acceptable salt thereof.
 59. The method of claim58, wherein the pharmaceutical composition is administered orally. 60.The method of claim 58, wherein the patient is a human.
 61. The methodof claim 58, wherein the pharmaceutical composition is in unit dosageform.
 62. The method of claim 61, wherein the unit dosage form comprisesabout 1 mg to about 400 mg of the compound or the pharmaceuticallyacceptable salt thereof.
 63. The method of claim 61, wherein the unitdosage form comprises about 5 mg to about 300 mg of the compound or thepharmaceutically acceptable salt thereof.
 64. The method of any one ofclaims 61 to 63, wherein the unit dose form is a capsule, a tablet, or apill.
 65. The method of claim 58, where the compound is administered asa crystalline freebase form.
 66. The method of claim 65, wherein thecrystalline freebase form is characterized by a powder X-ray diffractionpattern comprising diffraction peaks at 2θ values of 9.67±0.20,11.61±0.20, 17.61±0.20, 18.88±0.20, and 23.33±0.20.
 67. The method ofclaim 66, wherein the powder X-ray diffraction pattern further comprisesadditional diffraction peaks at 2θ values of 4.82±0.20, 15.69±0.20, and16.19±0.20.
 68. The method of claim 67, wherein the powder X-raydiffraction pattern further comprises two or more additional diffractionpeaks at 2θ values selected from 11.92±0.20, 12.98±0.20, 13.23±0.20,16.45±0.20, 16.67±0.20, 19.39±0.20, 19.96±0.20, 20.14±0.20, 22.14±0.20,23.84±0.20, 24.06±0.20, 24.29±0.20, 25.31±0.20, 25.63±0.20, 27.06±0.20,27.31±0.20, 30.10±0.20, and 30.53±0.20.
 69. The method of claim 65,wherein the crystalline freebase form is characterized by a powder X-raydiffraction pattern in which the peak positions are substantially inaccordance with the peak positions of the pattern shown in FIG.
 9. 70.The method of claim 65, wherein the crystalline freebase form ischaracterized by a differential scanning calorimetry trace recorded at aheating rate of 10° C. per minute which shows a maximum in endothermicheat flow at a temperature between 198° C. and 204° C.
 71. The method ofclaim 65, wherein the crystalline freebase form is characterized by adifferential scanning calorimetry trace recorded at a heating rate of10° C. per minute which shows a maximum in endothermic heat flow with apeak at 201.3° C.±2° C.
 72. The method of claim 65, wherein thecrystalline freebase form is characterized by a differential scanningcalorimetry trace substantially in accordance with that shown in FIG.10.